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Real-time PCR handbook

Single-tube assays

96- and 384-well plates

384-well TaqMan® Array cards

OpenArray® plates

Commonly used formats for real-time PCR.

Basics of real-time PCR 1
Experimental design 2
Plate preparation 3
Data analysis 4
Troubleshooting 5
Digital PCR 6

Basics of real-time PCR 1 .

10 Internal controls and reference genes 18 1.11 Real-time PCR instrument calibration 19 lifetechnologies.4 Real-time PCR analysis technology 6 1.6 Melting curve analysis 14 1.3 Overview of real-time PCR components 4 1 1.2 Overview of real-time PCR 3 1.5 Real-time PCR fluorescence detection systems 10 1.1 Introduction 2 1. Basics of real-time PCR 1.9 Multiplex real-time PCR 16 1.7 Passive reference dyes 15 1.com 1 .8 Contamination prevention 16 1.

amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. detection and quantification of the amplified 1 is measured by an instrument that combines thermal sequence are performed at the end of the reaction after cycling with fluorescent dye scanning capability. Relative fluorescence vs. you can achieve precise fluorescent signal in direct proportion to the number detection that is accurate within a 2-fold range. polymerase. Data dynamic range of input material covering 6 to 8 orders of collected in the exponential phase of the reaction yield magnitude. and thermal cycling. PCR product is measured at each the accumulation of product over the duration of the entire cycle. By plotting the last PCR cycle. heat-stable DNA that hybridize with PCR products during amplification. or dye molecules attached to PCR primers or probes using sequence-specific oligonucleotides. • An increased dynamic range of detection time quantitative PCR was developed. and involve post-PCR analysis such fluorescence against the cycle number. tube. with a of PCR product molecules (amplicons) generated. In traditional (endpoint) The change in fluorescence over the course of the reaction PCR. Over the past several years. In real-time instrument generates an amplification plot that represents quantitative PCR (qPCR). When it was first developed. users can determine the initial quantity of the target with great precision. amplification target. scientists reasoned that • Ability to precisely measure the amount the number of cycles and the amount of PCR end-product of amplicon at each cycle. Using these techniques. eliminating post-PCR manipulations cloning. By monitoring reactions during the exponential- PCR reaction (Figure 1). Basics of real-time PCR 1. many thousand. amplification phase of the reaction.1 Introduction The polymerase chain reaction (PCR) is one of the most quantitative information on the starting quantity of the powerful technologies in molecular biology. The advantages of real-time PCR include: PCR theoretically amplifies DNA exponentially. or “amplified”. real-time PCR has become In real-time PCR. The samples used to create the plots in this figure are a dilution series of the target DNA sequence.to a million-fold dyes. Fluorescent reporters used in real- specific sequences within a DNA or cDNA template can be time PCR include double-stranded DNA (dsDNA)–binding copied. doubling • Ability to monitor the progress of the PCR the number of target molecules with each amplification reaction as it occurs in real time cycle. endpoint • Amplification and detection occur in a single PCR is used mostly to amplify specific DNA for sequencing. 2 . cycle number. which allows could be used to calculate the initial quantity of genetic highly accurate quantification of the amount material by comparison with a known standard. To address of starting material in samples the need for robust quantification. the technique of real. Currently.   Figure 1. Amplification plots are created when the fluorescent signal from each sample is plotted against cycle number. Using PCR. and use in other molecular biology techniques. the amount of DNA is measured after the leading tool for the detection and quantification of DNA each cycle via fluorescent dyes that yield increasing or RNA. the real-time PCR as gel electrophoresis and image analysis. therefore.

this step is often combined with the annealing step. Real-time PCR steps One-step qRT-PCR There are three major steps that make up each cycle in a One-step qRT-PCR combines the first-strand cDNA real-time PCR reaction. using 60°C as the temperature. By measuring the amount using random primers. Reactions are generally run for 40 synthesis reaction and real-time PCR reaction in the cycles. To give an equal representation of all targets in 1 quantification is possible. oligo(dT). After reverse transcription. amplification is observed oligo(dT) primers. The denaturation time can be increased if template GC content is high. complementary sequences have an opportunity to hybridize. many researchers use random primers in earlier cycles. primers. so an appropriate temperature is used that is based on the calculated melting temperature (Tm) of the primers (typically 5°C below the Tm of the primer). Quantification of amplified product is obtained using fluorescent probes or fluorescent The temperature used for cDNA synthesis depends DNA-binding dyes and real-time PCR instruments that on the RT enzyme chosen.2 Overview of real-time PCR This section provides an overview of the steps involved in Two-step qRT-PCR performing real-time PCR. amplification or a mixture of oligo(dT) and random primers. (GSPs).com 3 . if the sequence is scarce. Real-time PCR is a variation Two-step quantitative reverse transcriptase PCR (qRT-PCR) of the standard PCR technique that is commonly used to starts with the reverse transcription of either total RNA or quantify DNA or RNA in a sample. measure fluorescence while performing the thermal approximately 10% of the cDNA is transferred to a separate cycling needed for the PCR reaction. 3. simplifying reaction setup and reducing the possibility of contamination. Extension: At 70–72°C. same tube. or gene-specific primers of amplified product at each stage during the PCR cycle. and primer extension occurs at rates of up to 100 bases per second. the activity of the DNA polymerase is optimal. Basics of real-time PCR 1. lifetechnologies. Denaturation: High-temperature incubation is used to are required. tube for the real-time PCR reaction. procedure and reduce the amount of product of interest. Annealing: During annealing. If a particular sequence (DNA or real-time PCR applications and to avoid the 3� bias of RNA) is abundant in the sample. the number of copies of a particular DNA or RNA This first-strand cDNA synthesis reaction can be primed sequence can be determined. is observed in later cycles. Using sequence-specific poly(A) RNA into cDNA using a reverse transcriptase (RT). This is because using oligo(dT) or random “melt” double-stranded DNA into single strands and primers will generate nonspecific products in the one-step loosen secondary structure in single-stranded DNA. The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). Gene-specific primers (GSP) 1. When an amplicon in real-time PCR is small. 2.

components like reporter dyes. high-quality dNTPs cDNA synthesis and may be important for accurate It is a good idea to purchase both the dNTPs and the mRNA quantification. and aseptic conditions should be as it is not uncommon to see a loss in sensitivity of one maintained. One option is to treat the background. The by using a “hot-start” enzyme. One of the main factors affecting PCR specificity solutions for each stage of the reactions.000 copies of template nucleic acid for each of qRT-PCR as the DNA polymerase. template with DNase I. Pure. To obtain tight data from synthesize nonspecific product. intact RNA is essential for full-length. Reverse transcriptase Template The reverse transcriptase (RT) is as critical to the success Use 10 to 1. This is equivalent to approximately choose an RT that not only provides high yields of full-length 100 pg to 1 μg of genomic DNA. The problem of nonspecific replicates (ideally. Basics of real-time PCR 1. In efficiency. prepare a master mix that products resulting from mis-priming can be minimized contains all the reaction components except sample. an RT that retains its activity at higher for cDNA rather than genomic DNA. The use of aerosol- at low temperatures. so enzyme selection is critical to technique. consequently. RNase contamination. A more detailed discussion of specific sulfate is typically used at a final concentration of 3 mM. but also has good activity at high temperatures. uracil DNA glycosylase (UDG) is provided in subsequent the optimal magnesium concentration may vary between sections of this handbook. allowing the polymerase to cross-contamination problems. however. RNA should be devoid of any thermostable DNA polymerase from the same vendor.3 Overview of real-time PCR components This section provides an overview of the major reaction Magnesium concentration components and parameters involved in real-time PCR In real-time PCR. full threshold cycle (Ct) in experiments that employ these isolation of mRNA is typically not necessary. or cDNA generated from cDNA. increasing specificity and reducing genomic DNA contamination. contamination and other pipetting errors. 3 and 6 mM. triplicates). reduces the chances of cross-well setup and the initial DNA denaturation step. from preparation is the fact that Taq DNA polymerase has residual activity of the template to post-PCR analysis. It is best to use dedicated equipment and success. and This concentration works well for most targets. Total RNA typically works well in qRT-PCR. it may be important to temperatures allows you to use a GSP with a high melting treat RNA templates to reduce the chance that they contain temperature (Tm). Depending on the specificity of the PCR primers one-step qRT-PCR. Excess template may also bring High-temperature performance is also very important higher contaminant levels that can greatly reduce PCR for denaturation of RNA with secondary structure. It is important to real-time PCR reaction. although it reagents from separate vendors. Using a hot-start enzyme use of a master mix reduces the number of pipetting steps ensures that DNA polymerase is not active during reaction and. Primers can anneal nonspecifically barrier tips and screwcap tubes can help decrease to DNA during reaction setup. 1 pg to 100 ng of total RNA. 1 DNA polymerase Good experimental technique PCR performance is often related to the thermostable Do not underestimate the importance of good laboratory DNA polymerase. passive reference dyes. 4 . may improve the yield of specific cDNAs. magnesium chloride or magnesium experiments.

Primer pairs should have compatible melting temperatures (within 1°C) and contain approximately 50% GC content. Analyze primer pair sequences to avoid complementarity and hybridization between primers (primer-dimers). Conversely. lifetechnologies.g. To confirm the specificity of your primers. poly(dG)) or repeating motifs. and be free of internal secondary structure. good primer design Primers should be designed according to standard PCR is especially critical when using DNA-binding dyes for guidelines. perform a BLAST® search against public databases to be sure that your primers only recognize the target of interest. Basics of real-time PCR Real-time PCR primer design Primer design software Good primer design is one of the most important parameters Primer design software programs. primers should be 18–24 nucleotides in length. As mentioned previously.. that primers are specific for the target sequence and free since longer products do not amplify as efficiently. keep in mind that 1 At a minimum.com 5 . For qRT-PCR. hybridization at 3� ends within each primer and with This provides for practical annealing temperatures. the 3� end of the primer should be GC rich to enhance annealing of the end that will be extended. Primers with high GC content can form stable imperfect hybrids. high AT content depresses the Tm of perfectly matched hybrids. A final concentration of 200 nM for each primer is effective for most reactions. If you choose to primers for it based on the criteria previously discussed. Optimal results may require a titration of primer concentrations between 50 and 500 nM. such as Vector NTI® Software. in addition to purchase TaqMan® Assay products—primers and probes sequence analysis software. using primer design software will ensure the amplicon length should be approximately 50–150  bp. such as OligoPerfect™ in real-time PCR. They should be specific for the target sequence amplicon detection. If possible. Primers should avoid stretches of homopolymer sequences (e. for real-time PCR designed using a proven algorithm and can automatically evaluate a target sequence and design trusted by scientists around the world. of internal secondary structure. and avoid complementary In general. design primers that anneal to exons on both sides of an intron (or span an exon/exon boundary of the mRNA) to allow differentiation between amplification of cDNA and potential contaminating genomic DNA by melting curve analysis. design your own real-time PCR primers. as these can hybridize inappropriately. This is why many researchers choose to designer and Primer Express® software. each other.

e.4 Real-time PCR analysis technology This section defines the major terms used in real-time PCR analysis. Threshold The threshold of the real-time PCR reaction is the level of signal that reflects a statistically significant increase over the calculated baseline signal (Figure 2). by user analysis or automated analysis of starting amount of the target template in experimental the amplification plot. will have a Ct one cycle earlier than that of the second sample.3 cycles apart. to allow accurate determination of the threshold cycle (Ct). The log of each known concentration in the dilution series defined below. but should not include the cycles in which the amplification signal begins to rise above background. real-time PCR instrument software automatically sets the threshold at 10 times the standard   deviation of the fluorescence value of the baseline. the baseline should be defined in the same way for each (Figure 2). For example. usually cycles 10-fold dilution series. This assumes that the PCR is operating at 100% efficiency (i. the Ct values are ~3. With a signal level during the initial cycles of PCR. Basics of real-time PCR 1. The baseline determination should take (x-axis) is plotted against the Ct value for that concentration into account enough cycles to eliminate the background found in the early cycles of amplification.. 3 to 15. in comparing real-time PCR results from samples containing different amounts of target. The low-level signal of the baseline can be equated to the Standard curve 1 background or the “noise” of the reaction (Figure 2). The baseline and threshold of a real-time PCR reaction. Ct (threshold cycle) The threshold cycle (Ct) is the cycle number at which the fluorescent signal of the reaction crosses the threshold. the position of the threshold can be set at any point in the exponential phase of PCR. Amplification plot for a 10-fold dilution series. Usually. It is set to distinguish relevant amplification signal from the background. 6 . However. a sample with twice the starting amount will yield a Ct one cycle earlier than a a sample with twice the number of copies of the target. Baseline As the template amount decreases. in which there is little change in fluorescent signal. because the Ct value is inversely related to the starting amount of target. The Ct is used to calculate the initial DNA copy number. the amount of product doubles perfectly during each cycle) in both reactions. The baseline should be set carefully samples or for assessing the reaction efficiency (Figure 4).   Figure 3. The A dilution series of known template concentrations can be baseline in real-time PCR is determined empirically for used to establish a standard curve for determining the initial each reaction. the cycle number at The baseline of the real-time PCR reaction refers to the which significant amplification is seen increases. Figure 2. When comparing different real- time PCR reactions or experiments. relative to a second sample.

y-intercept. To obtain accurate and reproducible results. which is present in a low amount.32. lifetechnologies.32 (see Efficiency.58 and –3. reactions should have an efficiency as close to 100% as possible. The concentrations chosen content of the amplicon can influence efficiency. This limits the Absolute quantification describes a real-time PCR usefulness of the y-intercept value as a direct measure of experiment in which samples of known quantity are serially sensitivity. all reagents are still in in one sample (i. the dynamic range for lowest copy number of target molecules denoted on the real-time PCR should be 7–8 orders of magnitude for x-axis gave rise to statistically significant amplification. treated) is compared to expression excess. All of these factors contribute to more accurate data. Slope The slope of the log-linear phase of the amplification reaction is a measure of reaction efficiency. Experimental parameters (including slope.999 is generally the maximum value. The presence of PCR inhibitors Correlation coefficient (R2) in one or more of the reagents can produce efficiencies The correlation coefficient is a measure of how well the of greater than 110%. or the Ct value expected if the in amplification product.e. the efficiency (E) of a PCR reaction should be 100%. and GC coefficient) can be derived. y-intercept. 0. Example of a standard curve of real-time PCR data.e. copy of a target. Basics of real-time PCR amplification product.. Figure 4. will not compete with the primers’ annealing capabilities. the DNA polymerase is still highly efficient. a copy number of 2–10 is commonly specified as the lowest target level that can be reliably Absolute quantification quantified in real-time PCR applications. secondary structure. which can result in efficiencies below 90%. A as determined by the following equation: standard curve shows threshold cycle (Ct) on the y-axis and the starting quantity of RNA or DNA target on the x-axis. below. for more detail).10. Other for the standard curve should encompass the expected conditions that may influence efficiency are the dynamics concentration range of the target in the experimental of the reaction itself. The R2 value reflects the efficiency between 90% and 110%. and the of the same gene in another sample (i. Ideally. Unknown samples are then quantified by comparison with this curve. Ideally. The actual efficiency can performance of the reaction as well as various reaction give valuable information about the reaction. although slope of between –3. plasmid DNA and at least a 3–4 log range for cDNA or Though PCR is theoretically capable of detecting a single genomic DNA. the use of non-optimal reagent samples. information about the during exponential amplification.com 7 . A good reaction should have an data fit the standard curve. Dynamic range Y-intercept This is the range over which an increase in starting The y-intercept corresponds to the theoretical limit of material concentration results in a corresponding increase detection of the reaction. Slope. meaning the template doubles after each thermal cycle (y-axis). untreated). However. Efficiency   A PCR efficiency of 100% corresponds to a slope of –3. Exponential phase It is important to quantify your real-time PCR reaction in Relative quantification the early part of the exponential phase as opposed to in the Relative quantification describes a real-time PCR later cycles or when the reaction reaches the plateau. and Efficiency = 10(–1/slope) –1 correlation coefficient values are used to provide information about the performance of the reaction. the y-intercept value may be useful for diluted and then amplified to generate a standard curve. comparing different amplification systems and targets. and correlation factors such as the length. equivalent to a 1 slope of –3. At the experiment in which the expression of a gene of interest beginning of the exponential phase. and enzyme quality.. Ideally. concentrations. which corresponds to a linearity of the standard curve. R2 = 1. From this standard curve.

For example. of each amplification reaction. Ct values are inversely related to the amount of starting template: the higher the amount of starting template in a reaction. This threshold is used to assign the threshold cycle. A normalizer gene (such as β-actin) is used as a control for experimental variability in this type of quantification. straightforward way to check real-time PCR reactions for primer-dimer artifacts and to ensure reaction specificity. Figure 6A illustrates a typical real-time PCR amplification plot. replicate consistency. amplicons) via melting curve analysis reduces the need for time-consuming gel electrophoresis.. The fluorescence is plotted against temperature (Figure 5A). GC content. in the case of relative quantification. against each other. when double-stranded DNA bound with SYBR® Green I dye is heated. and the presence of base mismatches. the level of fluorescence begins to increase with each cycle. The standard curve provides important information regarding the amplification efficiency. Ct values for a series of reactions containing a known quantity of target can be used to generate a standard curve. and theoretical detection limit of the reaction. The characterization of reaction products (e. As the reaction progresses. different PCR products can often be distinguished by their melting characteristics. During the early cycles of the PCR reaction. a sudden decrease in fluorescence is detected when the melting point (Tm) is reached. Melting curve (A) and –ΔF/ΔT vs. temperature (B). the lower the Ct value for that reaction. Basics of real-time PCR The results are expressed as fold change (increase or A decrease) in expression of the treated sample in relation to the untreated sample. 8 . there is little change in the fluorescent signal. primer- dimers vs.g. The reaction threshold is set above the baseline in the exponential portion of the plot. Quantification is performed by comparing Ct values for unknown samples against this standard curve or. among other factors. Melting curve (dissociation curve) A melting curve charts the change in fluorescence observed when double-stranded DNA (dsDNA) with incorporated dye 1 molecules dissociates (“melts”) into single-stranded DNA (ssDNA) as the temperature of the reaction is raised. The typical real-time PCR data set shown in Figure 6 Figure 5. illustrates many of the terms that have been discussed. Post-amplification melting-curve analysis is a simple. and then the –ΔF/ΔT (change in fluorescence/ change in temperature) is plotted against temperature to obtain a clear view of the melting dynamics (Figure 5B). with the standard curve serving as an efficiency check. Figure 6B shows the standard curve generated from the Ct values in the amplification plot. or Ct value. Because the melting temperature of nucleic acids is affected by length. due to dissociation of the DNA strands and subsequent release of B the dye.

25 x103 to 2 x10 4 copies. Real-time PCR of 2-fold serial dilutions of human RNase P DNA was performed using a FAM™ dye–labeled TaqMan® Assay with TaqMan® Universal Master Mix II. Basics of real-time PCR A 1   B   Figure 6. (A) Amplification plot. threshold cycle (Ct).com 9 . Amplification of RNase P DNA ranging from 1. under standard thermal cycling conditions on a ViiA™ 7 Real-Time PCR System. (B) Standard curve showing copy number of template vs. lifetechnologies.

However. If the probe is bound to the correct target sequence. the released reporter signal from the target and excludes signal from non-target molecule will no longer be quenched. The reporter signal is Figure 9. allowing the green dye to emit its full signal. FRET cannot occur. DNA polymerase extends the primer upstream of the probe. Basics of real-time PCR 1. the signal from the green dye will sphere represents a protein domain. If the red dye is in close proximity to the green dye. 1 The 5� nuclease domain has the ability to degrade DNA bound to the template. In FRET. While the probe is intact. releasing a fragment containing the reporter dye. transfer (FRET). the reporter and quencher have a natural affinity for each other.   allowing FRET to occur (Figure 11). (A) FRET occurs when a green light–emitting fluorescent dye is in close proximity to a red quenched prior to PCR. if the two dyes are not in close proximity. the polymerase’s 5� nuclease activity cleaves the probe. in close proximity. light–emitting fluorescent dye. but the most widely used are 5� nuclease assays such as TaqMan® Assays and SYBR® Green dye–based assays (Figure 7). downstream of DNA synthesis. but they are not 10 . the emissions of a fluorescent dye can be strongly reduced by the presence of another dye. The greatest threat to assay specificity for 5� nuclease assays is homologs. Specificity is arguably the most important aspect of any assay.5 Real-time PCR fluorescence detection systems Real-time fluorescent PCR chemistries Many real-time fluorescent PCR chemistries exist. the reporter and quencher dyes are Assay specificity is the degree that the assay includes no longer attracted to each other. In other words. FRET can be illustrated by two fluorescent dyes: green and red (Figure 9). The 5� nuclease assay is named for the 5� nuclease activity associated with Taq DNA polymerase (Figure 8). A representation of Taq DNA polymerase. energy is being transferred from a higher to a lower   Figure 8. Once 5� nuclease assay specificity cleavage takes place. in the results. the TaqMan® probe is intact and has a degree of flexibility. be suppressed or “quenched”. often called the quencher. the primers and probe anneal to the target. Homologs are genes similar in sequence to that of the target. (B) FRET does not occur when the two fluorescent dyes are not in close proximity. Each colored level. A second key element in the 5� nuclease assay is a phenomenon called fluorescence resonance energy Figure 7. excitation of the green dye will cause the green emission energy to be transferred to the red dye. Before PCR begins. Example of the FRET phenomenon. Consequently. During PCR. The green fluorescent dye has a higher energy of emission compared to the red. because green light has a shorter wavelength compared to red. A 5� nuclease assay for target detection or quantification typically consists of two PCR primers and a TaqMan® probe (Figure 10). Representation of a 5� nuclease assay (left) and SYBR® Green   dye binding to DNA (right).

TAMRA™ probe. the intended target of the assay. At that point. 5� nuclease assays offer two tools for specificity: primers and probes. PCR may also amplify nonspecific greater impact on TaqMan® MGB probe binding. They used a dye called TAMRA™ dye as the TaqMan® MGB probe. A mismatch between the target and homolog positioned at the 3�-most base of the primer has maximal TaqMan® probe types impact on the specificity of the primer. which is a fluorescent dye that will report the amplification of the target. A mismatch further TaqMan® probes may be divided into two types: MGB and away from the 3� end will have less impact on specificity. so that instead of can be much shorter than PCR primers.com 11 . signal is enables TaqMan® MGB probes to bind to the target more produced from the target but not from the homolog. was desirable. However. Representation of a TaqMan® probe in solution. and the orange line 1 primers. Early in the development of real-time PCR. a short minor groove probe binding site cannot be stabilized by the DNA is formed in the DNA. can have a strong impact on specificity— extensive testing revealed that TaqMan® probes required TaqMan® MGB probes are stronger tools for specificity an annealing temperature significantly higher than that than primers. On the 3� end of the probe is a quencher. Since nonspecific products high genetic complexity. the intact probe is displaced. The intact probe MGB moiety. the shorter probe size. even specifically than primers at higher temperatures. TaqMan® probes were therefore longer than primers. a 1-base mismatch has a much In addition to homologs. The first TaqMan® probes could be classified In contrast. of PCR primers to allow cleavage to take place. a higher degree of specificity so it is just as stably bound as primer bound to the intended. mismatches across most of the length of a as non-MGB. TaqMan® MGB probes possess a minor-groove binding (MGB) molecule on the 3� end. Because of the cleavage. The quencher also blocks the 3� end of the probe so that it cannot be extended by thermostable DNA polymerase. thus strengthening Mismatches in a TaqMan® MGB probe binding site will probe binding. And products produced by primers binding to seemingly random because of this higher level of specificity. allowing cleavage to take place. probe binding sites. TaqMan® MGB locations in the sample DNA or sometimes to themselves probes are recommended for most applications involving in so-called “primer-dimers”. so that when probes and still achieve a high melting temperature. TaqMan probe. which quenches fluo- rescence from the reporter in intact probes. allowing the MGB molecule to bind polymerase due to the quencher block on the 3� end. with high efficiency. Homologs are extremely are unrelated to the target. they do not have TaqMan® common within species and across related species. there is nothing to prevent the DNA polymerase from continuing synthesis TaqMan® MGB probes were a later refinement of the to produce a copy of the homolog. and thus are not seen in the real-time PCR data. Consequently. non-MGB. mismatches on the 5� end of the TaqMan® Where the probe binds to the target.or 2-base random mismatch in mismatch in such long probes had a relatively mild effect the primer binding site will very likely allow the DNA on probe binding. Attached to the 5� end of the TaqMan® probe is the “reporter”. these probes can be shorter than TaqMan® returns to its quenched configuration. In contrast. TaqMan® MGB probes reduce how tightly the probe is bound. A 1-base For example. represents the oligonucleotide. A one or two base extension by DNA such as eukaryotic gene expression and single nucleotide polymerase will stabilize the primer bound to the homolog. fully complementary target. polymerase to extend the primer bound to the homolog for many applications involving high genetic complexity. polymorphisms (SNPs). and increase the melting temperature. The TaqMan probe has a gene-specific ® ® Figure 11. This data are collected at the end of the PCR cycle. lifetechnologies. TaqMan® probe technology. Basics of real-time PCR     Figure 10. a 1. Q is the quencher molecule. R is the sequence and is designed to bind the target between the two PCR reporter dye. With though the homolog is being amplified. which is shorter than a TaqMan® quencher.

target or non-target. such as shoulders. dye. from the baseline. or splits. casting strong signals are summed. analysis. is one defined genetic sequence. nor one Since amplification of non-target can vary from sample product. automatically subtracted to zero in the assessment because it does not add cost to the experiment real-time PCR software. may suppress the amplification of the target or. is termed “baseline”. If the sample and can be done right in the PCR reaction vessel. values (e. whether the amplification consists of target. but Tm and shape with a particular target after amplification. one. non-target. Under ideal conditions. Dissociation is an attractive choice for specificity This early signal. the researcher will be able to associate a peak Assay specificity testing is important for all assays. it should have one specific melting temperature (Tm). Wells with SYBR® Green amplification plot shape cannot be used to dissociation anomalies should be omitted from further assess specificity. making them more vulnerable to anomalies. and devoid of other of a TaqMan® probe. If the target was present amplification of pure target. A SYBR® Green dye–based assay typically consists of Following the melt process. allowing detection of those non-target amplifications. 1 becomes visible is inversely related to the initial target The concept of SYBR® Green dissociation is that if the target quantity. and all such of similar Tm values were produced. a Accurate identification of the target peak depends on horizontal baseline is observed. In the early PCR PCR products after PCR when using SYBR® Green–based cycles. both follow SYBR® Green dissociation is the gradual melting of the the same pattern for signal production. Therefore. The point at which amplification signal and has limited resolution. The fact that a SYBR® Green assay produced an SYBR® Green dissociation is low resolution and may not amplification should not be automatically taken to mean differentiate between target and non-target with similar Tm the majority of any of the signal is derived from target. especially for those most vulnerable to specificity problems. contains a target. doubts about the specificity of those reactions. eventually enough of the cleaved probe dissociation does add more time to the thermal protocol.g. Plots usually have the same appearance. will accumulate to allow amplification signal to emerge requires additional analysis time. By starting with SYBR® Green assay specificity pure target. humps.. in some cases. which assay follows an amplification pattern similar to that of a transforms the melt profile into a peak. to sample. a SYBR® Green will plot the data as the negative first derivative. Most Dissociation data should be evaluated for each well where commonly. without additional supporting information. which is used to help identify the SYBR® Green dye target in samples. The presumptive target SYBR® Green assays do not benefit from the specificity peak should be narrow. narrow symmetric peak should not be assumed to be the target. detection. TaqMan® probe–based assay. These specificity problems. the 12 . is somewhat subjective. If the sample contains a peak analysis. SYBR® Green dye will bind to any anomalies are strong indications that multiple products amplified product. binds to the minor groove of any double-stranded DNA. only the low. Some non-target products will have SYBR® Green I dye is a fluorescent DNA-binding dye that Tm values significantly different from that of the target. be produced at some point so that amplification signal creating opportunities for non-target amplification that becomes visible. Many samples such as cellular in the sample. at least one type of specificity assessment should be performed for every SYBR® Green reaction. that does not correspond to the pure target peak. Only one peak should be observed. quenched reporter signal is detected. However. alter the shape of the melt peak. producing a single amplification plot. sufficient accumulated PCR product will RNA and genomic DNA exhibit high genetic complexity. homologs). Basics of real-time PCR TaqMan® probe signal production SYBR® Green dye dissociation Whether an MGB or non-MGB probe is chosen. the real-time PCR software two PCR primers. this ongoing assessment is the dissociation amplification was observed. or a mixture. In the early PCR cycles. symmetrical. Excitation of DNA-bound SYBR® Green dye produces a much stronger fluorescent signal compared to unbound The dissociation protocol is added after the final PCR cycle.

target may have amplified in that reaction. as in the ViiA™ 7 system. a passive reference dye. 384-microwell cards. and a detector to detect the fluorescent emissions. either through optimized combinations of excitation and emission filters or through a process called multicomponenting. etc. or gels. Multiple dyes in the same well can be measured independently. the QuantStudio® 6 and 7 Flex systems. All real-time PCR instruments also come with software for data collection and analysis. Multicomponenting is a mathematical method to measure dye intensity for each dye in the reaction. sequencing. 1 Real-time PCR instrumentation Many different models of real-time PCR instruments are available. but when combined with other information. in some cases a quencher dye. refreshing optical performance to factory standard without hardware adjustment. 3. including one or more reporter dyes. and. If the sample contains a peak that appears to match the Tm and shape of the pure target peak. 96-well plates. as in the StepOnePlus™ system or user interchangeable. Blocks are available to accept a variety of PCR reaction vessels: 48-well plates. which excites the fluorescent dyes. each model must have a thermal cycler. Basics of real-time PCR conclusion is that target was not detected in that reaction. The thermal block may be either fixed. such as data from target-negative samples. Dissociation data in isolation cannot be taken as definitive. Multicomponenting offers the benefits of easy correction for dye designation errors.com 13 .072–through-hole plates. In addition. Dye differentiation Most real-time PCR reactions contain multiple dyes. and provides a source of troubleshooting information. 384-well plates. can provide more confidence in specificity. Each model must have an excitation source. very often. and QuantStudio® 12K Flex system. lifetechnologies.

a decrease of NTCs can discriminate between primer-dimers and in fluorescence will be seen as soon as the DNA becomes spurious amplification due to contaminating nucleic acids single-stranded and the dye dissociates from the DNA. may form primer-dimers or amplify a nonspecific product After amplification is complete. If there are primer-dimers in the NTC. however. data (Figure 13). the increased specificity is an abundance of primer and no template. Most real-time PCR instrument platforms which may also be amplified. the instrument will reheat (Figure 12).6 Melting curve analysis Melting curve analysis and detection Melting curve analysis and primer-dimers systems Primer-dimers occur when two PCR primers (either Melting curve analysis can only be performed with real. GreenER™ dyes significantly increases upon binding to the primers should be redesigned. There is also the possibility when performing your amplified products to give complete melting curve qRT-PCR that the RNA sample contains genomic DNA. the real-time PCR the primers and reaction conditions used. there instrument can be programmed to include a melting is always the possibility that even well-designed primers profile immediately following the thermal cycling protocol. The formation of primer-dimers most in signal by cleaving and releasing the fluorophore into often occurs in no-template controls (NTCs). Basics of real-time PCR 1. Importance of melting curve analysis How to perform melting curve analysis The specificity of a real-time PCR assay is determined by To perform melting curve analysis. The presence of this method makes this less of a concern.   Figure 12. such as primer-dimers. as it decreases PCR efficiency and compatible because they produce an irreversible change obscures analysis. DNA. However. 14 . By monitoring the dsDNA as it melts. as shown by the Applied Biosystems® instrument (rapid heating to 94°C to denature the additional peaks to the left of the peak for the amplified product peaks. of primer-dimers in the NTC should serve as an alert to the user that they may also be present in reactions that The level of fluorescence of both SYBR® Green I and SYBR® include template. When melting curve analysis is not possible. Melting curve analysis dsDNA. same-sense primers or sense and antisense primers) bind time PCR detection technologies in which the fluorophore to each other instead of the target. Amplifications can identify the presence of primer-dimers because they that have used SYBR® Green I or SYBR® GreenER™ dye exhibit a lower melting temperature than the amplicon. Melting curve analysis can detect the presence of Figure 13. 1 can be subjected to melting curve analysis. followed by cooling to 60°C). where there solution during the PCR. Melting curve analysis remains associated with the amplicon. now incorporate this feature into their analysis packages. in the reagent components. Example of a melting curve thermal profile setup on an nonspecific products. The specificity of the real. Dual-labeled The presence of primer-dimers is not desirable in samples probe detection systems such as TaqMan® probes are not that contain template. additional care must be used to establish that differences observed in Ct values between reactions are valid and not due to the presence of nonspecific products. time PCR reaction can be confirmed using melting curve analysis.

Well factors for for fluctuations from well to well caused by changes experiments using SYBR® Green I or SYBR® GreenER™ dye in reaction concentration or volume and to correct for are calculated using an additional fluorophore. A passive reference dye such as ROX™ dye is used to normalize the fluorescent reporter signal in real- time PCR on compatible instruments. which uses fluorescein as the reference dye. which can then be Passive reference dye referenced for future readings. Most real-time PCR 1 instruments use ROX™ dyes as the passive reference Well factors are collected using either a separate plate dye. The iCycler® iQ™5 and MyiQ™ systems allow you to save the data from an external well factor reading as a separate file.g. variations in instrument scanning. The use of a passive reference dye is an effective tool for the normalization of fluorescent reporter signal without modifying the instrument’s default analysis parameters.com 15 . Normalization is necessary to correct instrument or pipetting non-uniformity. caused by variation in pipetting) • Normalize for fluctuations in fluorescence resulting from machine “noise” • Compensate for variations in instrument excitation and detection • Provide a stable baseline for multiplex real-time PCR and qRT-PCR lifetechnologies. fluorescein. because ROX™ dye does not affect the real-time containing fluorescein dye in each well (external well PCR reaction and has a fluorescent signal that can be factors) or the experimental plate with fluorescein spiked distinguished from that of many reporter or quencher dyes into the real-time PCR master mix (dynamic well factors). such as Applied Biosystems® instruments.7 Passive reference dyes Passive reference dyes are frequently used in real-time Fluorescein reference dye PCR to normalize the fluorescent signal of reporter dyes Bio-Rad iCycler® instruments require the collection of and correct for fluctuations in fluorescence that are “well factors” before each run to compensate for any not PCR-based. TaqMan® real-time PCR master mixes contain a passive reference dye that serves as an internal control to: • Normalize for non–PCR-related fluctuations in fluorescence (e.. Basics of real-time PCR 1. An exception is the Bio-Rad iCycler iQ® instrument You must select the method when you start each run system. using the iCycler® instrument. used.

detection of two or more genetic sequences in the same reaction. PCR multiplexing must be able Multiplexing benefits to produce sufficient amplified product for the detection of Three benefits of multiplexing—increased throughput all of the intended sequences. Target and normalizer data from the same well are derived from a single sample addition. will increase electrophoresis can be used. it may be possible to combine two target an assay designed to amplify a single genetic sequence. all of the intended on the number of targets in the experiment. preventing its use as a template in PCR. and amplify two genetic sequences. Comparing multiplex data analyzed in a to multiplex and is responsible for multiplex validation. by preventing the amplification of DNA from previous A heat-labile form of the enzyme is also available. Uracil N-glycosylase (UNG) The removal of the uracil bases causes fragmentation of Uracil N-glycosylase (UNG) is used to reduce or prevent the DNA. For example. PCR reaction and the reliability of data. the MicroSEQ® normalizer results equally. For qualitative results. The use of UNG in PCR reactions reduces false is inactivated at 50°C. another benefit of multiplexing is minimizing pipet possible. so that it can be used in one-step positives. • Cross-contamination between samples which degrades any contaminating uracil-containing PCR • Contamination from laboratory equipment products. such as an endogenous control assay. sample usage. In order to gain this precision E. Basics of real-time PCR 1. For research data from the same well before calculating technical applications. Singleplex is target assays. 1 • Carryover contamination of amplification products and primers from previous PCRs. in which the assay for the target gene is conducted in the same well as that for the control If the target assay is multiplexed with the normalizer or normalizer gene. of multiplex is a duplex. To be successful.9 Multiplex real-time PCR Introduction to multiplexing important to weigh the benefits of multiplexing versus the PCR multiplexing is the amplification and specific degree of effort needed for validation. if amplified products are running the target assay as a duplex with the normalizer sufficient an endpoint detection method such as gel assay. assays and the normalizer assay in a triplex reaction. The DNA carryover contamination between PCR reactions UNG is then inactivated in the ramp up to 95°C in PCR. it is analysis done in a multiplex manner demonstrates that the 16 . Real-time PCR multiplexing (more samples potentially assayed per plate). If a quantitative experiment consists of two The suffix “plex” is used in multiple terms. target data must be normalized by the normalizer with an internal positive control assay. Subsequent contamination are: real-time PCR reaction mixes are then treated with UNG. the scientist usually chooses which assays replicate precision. but higher-order multiplexes are also assay. precision errors. and reduce reagent usage by half. the throughput increase. throughput. reduce sample required. a short incubation at 50°C is performed to be the major source of false positive PCR results prior to the PCR thermal cycling so that the enzyme can cleave the uracil residues in any contaminating DNA. so Some commercial real-time PCR kits are designed and any pipet precision error should affect both the target and validated as a multiplex. if sequences must produce sufficient geometric-phase a quantitative experiment consists of only one target assay. This is considered With standard UNG. in turn increasing the efficiency of the real-time qRT-PCR reaction mixes. which reactions. The most common type reagent reduction will be even greater. signal.8 Contamination prevention As with traditional PCR. leaving the natural (thymine-containing) target DNA template unaffected. leading to UNG for carryover prevention begins with the substitution of false positive results. In Duplex is a combination of two assays designed to that case. sample reduction. coli target assay benefit. Some of the possible sources of dUTP for dTTP in real-time PCR master mixes. real-time PCR reactions can How UNG carryover prevention works be affected by nucleic acid contamination. coli O157:H7 Kit multiplexes the E. For example. reduced may be used to produce quantitative or qualitative results. singleplex manner (without well-based normalization) to an When considering whether to create a multiplex assay. 1. and reduced reagent usage—are dependent For quantitative PCR multiplexing.

Generally. such as ABY® or JUN® dye. Whereas blue-only excitation abundance in all samples. For example. The 18S rRNA detected in the multiplex will require a different reporter dye. the two genes have approximately equal a red passive reference dye. The vast amplification. high. multiple duplex assays may be created by Multiplexing is one recommended method to help achieve pairing a different FAM™ dye–labeled target assay with the the necessary degree of precision for this type of experiment. this pattern. a Ct difference between the two genes of 3 or higher. Note that if ABY® dye is being Multiplex assays usually involve multiple dyes in the same used. suppressing the amplification of the less Chemistry recommendations for abundant gene. the normalizer might be 18S ribosomal chemistry is being used. with accuracy. each genetic sequence being RNA. well. benefit will be minimal as well. Reporter dyes do not have to be assigned based on the type The precision benefit of multiplexing is especially valuable of target gene or gene product. of precision. FAM™ dye as the reporter for the target assay and assigning 1 However. FAM™ dye is the most common reporter dye experiments. exhausted before the PCR product accumulates to a level specificity chemistries. the three most primer concentrations optimal for multiplexing. When planning a multiplex assay. Only the assay Dye choices for multiplexing for the more abundant target requires primer limitation. two-step RT-PCR is identify which gene or genes in the multiplex have the generally recommended over one-step RT-PCR. termed “primer limitation”. of the more abundant gene saturates the thermostable DNA polymerase. In a triplex assay. The remedy for saturation is a reduction multiplexing of the PCR primer concentration for the more abundant The best fluorescent chemistries for real-time PCR target. These measurements must remain specific for each dye. assuming the same lifetechnologies. RT-PCR. which requires even better precision. The instrument manufacturer should be able to offer dye recommendations. Using 1. depending instruments can excite this ROX™ dye–based reference on the singleplex error.com 17 . discriminating 2 copies from 3 copies is only a VIC® dye as the reporter for the normalizer assay. for samples with sufficiently to act as a passive reference dye. assays. In this regard. the more abundant gene or gene product is the same in all samples. real-time PCR instrument. but following a pattern in for quantitative experiments requiring a higher degree assigning dyes can simplify the creation of a multiplex assay. The primer-limited multiplexing are those that can assign different dyes to concentration should be sufficient to enable geometric detect each genetic sequence in the multiplex. Therefore. One-step potential to cause saturation. In this most common scenario. only the 18S assay would require accurately detected when together in the same well by the primer limitation. the researcher should For multiplex assays involving RNA. may be combined Instrumentation for multiplexing with FAM™ dye and VIC® dyes. reducing flexibility in product in the PCR reaction. MUSTANG PURPLE® dye should measuring those different dye signals in the same well be used instead of ROX™ dye as a passive reference. which is 20% of eukaryotic total RNA.5-fold difference. such as TaqMan® probe-based that starves amplification of the less abundant target. the PCR primer concentration may be optimized for multiplexing. same VIC® dye for the normalizer assay. TAMRA™ dye should not be present in the same well. even when one dye signal is significantly Multiplex PCR saturation higher than another. Proper instrument calibration is Multiplex PCR saturation is an undesirable phenomenon necessary to accurately measure each dye contribution that may occur in a multiplex assay when the amplification within a multiplex assay. blue excitation minimal singleplex precision error. For example. but sufficiently low that the primer is majority of multiplexing is performed with multi-dye. Basics of real-time PCR multiplex precision benefit can be substantial. the multiplex precision is generally not sufficient for red dyes to act as a reporter. Note that Duplex scenario 2 Applied Biosystems® real-time PCR master mixes contain In this scenario. Assuming a multi-dye real-time PCR fluorescence For example. without having any adverse affect on Duplex scenario 1 reverse transcription. discriminating 1 copy from 2 copies of the used in TaqMan® probes. The real-time PCR instrument must be capable of and if JUN® dye is used. In two-step common duplex scenarios are listed below. in copy number variation For example. a third dye. We follow the pattern of assigning gene is a 2-fold difference. cDNA would be more abundant than any mRNA cDNA in The reporter dyes chosen must be sufficiently excited and every sample. which is based on the RT-PCR requires the same primer concentration for absolute DNA or cDNA abundance of each gene or gene reverse transcription and PCR. which requires good precision.

For example. the expression level of the internal standard (or endogenous control) should be the chosen housekeeping gene should be validated for expressed at roughly the same level as the experimental each target cell or tissue type to confirm that it remains gene product. For example. To achieve accurate and reproducible housekeeping genes expression profiling of selected genes using real-time Relative gene expression comparisons work best when the PCR.10 Internal controls and reference genes Real-time PCR has become a method of choice for gene Relative gene expression analysis using expression analysis. interactions can create competitive products. Multiplex primer interactions Another potential threat to multiplex assay performance is unexpected primer interactions between primers from different assays. of significantly different Ct values in the singleplex vs. it is critical to use reliable internal control gene expression level of the chosen housekeeping gene remains products for the normalization of expression levels constant. are duplex assay with 4 PCR primers there are 6 unique primer most likely to fall into this scenario. The chances of primer interactions grow Duplex scenario 3 when the assays being multiplexed have homology. The risk of primer interaction grows with the number of assays in the reaction. quantification of an mRNA target can GAPDH expression has been shown to be up-regulated in be normalized for differences in the amount of total RNA proliferating cells. The target chosen to be Endocrinol 25:169 (2000). suppressing amplification. Basics of real-time PCR threshold. In singleplex each assay progressing through the geometric phase at approximately may perform well. in a DNA applications. Genomic the number of assays in the multiplex. 18 . and in a triplex assay with 6 PCR primers not necessary for scenario 2. chosen to act as the endogenous control. but in a multiplex reaction the primer the same time. the remedy is to use a different assay in the multiplex reaction. because the number of unique primer pairs increases dramatically with 1. The choice of the housekeeping reference gene between experiments—typically expression products from is reviewed in BioTechniques 29:332 (2000) and J Mol housekeeping genes are used. In this case. because the two genes are there are 15 unique pairs possible. both assays should be primer limited. active reference. Regardless of the gene that is always represent the overall cellular mRNA population. such as copy number variation. Ideally. By using an endogenous control as an constant at all points of the experiment. that gene must be tested under all of one’s experimental conditions. Primer limitation is pairs possible. either the gene or gene product has the primer interaction does occur based on the observation 1 potential to be significantly more abundant than the other. would not qualify for equal abundance. multiplex reaction. If In this scenario. and 18S ribosomal RNA (rRNA) may not added to each reaction. to ensure that there is consistent expression of the control gene under all conditions.

so the resulting corrected Second. the phenomena occur. At that high properly. such as how the reagents and plate were prepared. heat and pressure of the heated lid causes the seal to seat During cycling. With termed “optical warping”. which may be above the liquid. A second example is large bubbles that burst temperature. if a passive reference dye is present degree of improvement will vary. Optical warping occurs when this configuration. and the emission detector. creating small air Normalization does not fully correct for a large bubble bubbles. do affect the precision of replicates. It preserves plastic seal. to the reagents at the bottom. This variation may increase with Precision improvement age and usage of the instrument. following the manufacturer’s instructions.com 19 . and then. causing it to change shape slightly during data integrity and consistency over time. those differences will affect the reporter factors. air dissolved within the liquid amplification plot may appear completely anomaly-free. emission sensitivity across the wells of the block. correction for optical warping. air space is configuration change in the plate seal. a number of temperature-related a well is inadequately sealed. and passive reference signals to the same degree. Basics of real-time PCR 1. Uncorrected excitation/ The correction effect of passive reference normalization emission differences across the plate can cause shifts in will improve the precision of real-time PCR data. is continuous during PCR. Atypical optical fluctuations Atypical optical fluctuations are thermal-related anomalies that are not universal across all reactions in the run and Universal optical fluctuations produce an obvious distortion in the reporter signal. accurate calibration is critical for the proper Third. lifetechnologies. well. a passive such as halogen lamps or LEDs. at high temperature. This entire process. depending on factors such as how Excitation/emission difference much air was dissolved in the reagents and how well the corrections plate was sealed. but they be divided into two main categories: the excitation source.11 Real-time PCR instrument calibration Timely. bursting. called Normalization to a passive reference dye provides excellent “refluxing”. One In traditional plastic PCR plates and tubes. If present. This water vapor or steam will condense on the Distortions in the amplification plot are likely to cause cooler walls of the tube. The Ct values. and a plastic seal is over the well. depending on a number of in the reaction. forming water droplets that return baseline problems and may even affect Ct values. the liquid example of an atypical optical fluctuation is a significant reagents are at the bottom of the well. reagents will become less soluble. but it can help minimize the data distortion caused. Real-time PCR PCR. during PCR. instruments should be calibrated as part of a regular All of these temperature-related phenomena are in maintenance regimen and prior to using new dyes for the the excitation and emission light path and can cause first time. such as a CCD camera or photodiode. Generally. However. fluctuations in fluorescent signal. the pressure of the steam will exert force on the performance of any real-time PCR instrument. temperatures reach 95°C. The degree of these 1 fluctuations can vary. water is volatilized into the air space in the during PCR. reference dye is in the same light path as the reporter. so that normalization of the reporter to the passive reference corrects the difference. While manufacturers so normalization of reporter to passive reference signals can achieve excellent uniformity for excitation strength and corrects for these fluctuations. universal fluctuations do not The optical elements in real-time PCR instruments can produce obvious distortions in the reporter signal. there will always be some variation.

Experimental design 2 .

6 Controls 30 2.5 Reverse transcription considerations 28 2.4 Nucleic acid purification and quantitation 26 2 2. and reproducibility 32 lifetechnologies. Experimental design 2.com 21 . sensitivity.1 Introduction 22 2.3 Amplicon and primer design considerations 23 2.8 Using a standard curve to assess efficiency.7 Normalization methods 30 2.2 Real-time PCR assay types 22 2.

and guidelines for during this process. Often.2 Real-time PCR assay types Gene expression profiling is a common use of real-time PCR that assesses the relative abundance of transcripts is for counting functional viral particles or the total number of particles. what • Nucleic acid purification biological questions need to be answered. endpoint fluorescence is measured to lents or nucleic acid harvested from a titered virus control. After determining your experimental goal. reverse transcription efficiency. Assay design focuses on real-time PCR efficiency and the Viral titer determination assays can be complex to design. Up-front planning will implementation. the genome is analyzed RNA quality. In copy number variation analysis.1 Introduction Successful real-time PCR assay design and development describes the stages of real-time PCR assay design and are the foundation for accurate data. different from one designed to determine viral copy number and reproducibility from that same disease state. determine the SNP genotypes. The assay design. an • Reverse transcription experiment designed to determine the relative expression • Controls and normalization level of a gene in a particular disease state will be quite • Standard curve evaluation of efficiency. researchers want to quantify viral copy number in Lastly. assist in managing any experimental variability observed the role they play in data accuracy. Depending on the nature of allele-specific cross-reactivity. Assay design will also be influenced by whether the assay 22 . This section 2 2. will be dictated normalizer gene play particularly important roles in gene by whether relative or absolute quantification is desired. and most PCR efficiency. clearly • Target amplicon and primer design understand the goal of the assay. Experimental design 2. allelic discrimination assays can detect variation samples. We will identify sources of variability. This is often accomplished by comparison to a down to the single-nucleotide level. of the target—an RNA or DNA virus—reverse transcription and real-time PCR efficiency also play significant roles. and the choice of a specifically standard curve generation. expression experiments. Unlike the methods standard curve generated using known genome equiva- described above. identify the appropriate real-time PCR controls and opportunities for optimization. Primer and probe design Success is dependent on the accuracy of the material used play particularly important roles to ensure a low incidence to generate the standard curve. sensitivity. real-time for duplications or deletions. For example. optimization in the following areas: Before embarking on experimental design. accuracy necessary to discriminate single-copy deviations. quantification strategy. to determine gene expression patterns between samples. specifically.

Thus. primers anneal within the same exon. primers to be sure that their binding sites are unique in reaction efficiency is paramount to the accuracy of real. structure obstructs the path of the DNA polymerase. any eliminating primer designs in these locations. For this reason. deviation from 100% efficiency can result in potentially erroneous data. Less-than-perfect target The best method for avoiding gDNA interference in real- doubling at each cycle is more likely to occur if secondary time PCR is thoughtful primer (or primer/probe) design. Primer design software programs can perform BLAST® searches to avoid pseudogenes and their mRNA products. This reduces the possibility that the primers time PCR data. Primer sets for SYBR® locate amplicons near the 3� ends of transcripts. Genomic DNA. it is best to spans an exon-exon boundary. In detected by setting up control reactions that do not contain addition. These are derivatives of existing genes that have become nonfunctional due to mutations and/or rearrangements in the promoter or gene itself. When upstream and downstream PCR likely to be impacted (Figure 14). doubling the genome. In a perfect scenario. primers should be designed to anneal with. these amplicons are less exon/exon junction. Pseudogenes. Conversely. An RNA molecule with a high degree of secondary structure. upstream target. a region of medium (50%) GC content with no Expression Assays are designed so that the TaqMan® probe significant GC stretches. For amplifying cDNA. it indicates that gDNA is not contributing to signal and downstream primers will be less likely to find their generation. and are absent in mRNA. Amplifying a 100 bp region is much more 2 concern when measuring gene expression levels. Variations in efficiency originating genome and masking homologous areas. amplifying a 1. say. gDNA can compromise the efficiency complementary sequence in the same DNA fragment. Primer design software programs automate number of full-length target molecules: this corresponds the process of screening target sequences against the to 100% amplification efficiency. When designing real-time PCR primers. However. only cDNA will be amplified in most cases. lifetechnologies. Target specificity is another important factor in data location. Figure 14. thus will be amplified as thermal cycling progresses. and specificity accuracy. TaqMan® Gene to amplify. Whenever possible. resulting in an amplicon that is too long to amplify efficiently in the conditions used for real-time PCR. Experimental design 2. Genomic DNA contamination is real-time PCR target lengths are generally 60–200 bp. which takes advantage of the introns present in gDNA that Ideally. if the Ct for the RT in template integrity.200 bp target. If RNA Green dye–based detection should be designed to anneal secondary structure prohibits full-length cDNA synthesis in adjacent exons or with one of the primers spanning an in a percentage of the transcripts. If nucleic acid samples are slightly control is higher than the Ct generated by the most dilute degraded and the target sequence is long. GC content. resulting in invalid data. when primers anneal in adjacent exons. than.com 23 . of the reaction due to competition for reaction components Amplicon GC content and secondary structure can be such as dNTPs and primers. pseudogenes. are other transcript variants to consider when designing primers. and allele One way to minimize efficiency bias is to amplify relatively variants Genomic DNA carryover in an RNA sample may be a short targets. shorter amplicons are less affected by variations reverse transcriptase (RT control). the genome. The gDNA likely to result in complete synthesis in a given cycle may be co-amplified with the target transcripts of interest. or silent genes. because the amplicon from gDNA would include intron sequence. another cause of data inaccuracy.3 Amplicon and primer design considerations Target amplicon size. check As will be discussed in more detail later in this guide. they can amplify target from both DNA and RNA. each target copy in could amplify similar sequences elsewhere in the sample a PCR reaction will be copied at each cycle.

as often is the case. but can also be secondary structure within primers (which could lead to individual 2 the result of forward-forward or reverse-reverse primer primers folding on themselves) or stretches of sequence that would allow primers to anneal to each other. Specificity. In addition. One Assays). the lower the amount of target at the start of the PCR reaction. tion. which can be incorrectly diagnosed as off-target amplification. However. dimers appear as diffuse bands at the bottom of the gel. If the dimerization occurs in a staggered manner. is that dimerization experimentally. depending on whether one or more variants are being studied. the more likely primer-dimer formation will be. for detecting primer-dimers. Another problem is that the resulting multiplexing applications. This software Primer-dimers are most often caused by an interaction is used to analyze primer sequences and report areas of potential between forward and reverse primers. and there are many ways to minimize serially diluted and added to the components of a PCR mix. primer-dimers themselves (as is the case with TaqMan® smudgy bands near the bottom of the gel (Figure 16). also related to efficiency. Primer- or eliminate this phenomenon. Even if signal is not generated from the is gel electrophoresis. This is especially helpful with SYBR® Green I dye. the dynamic range of the reaction may shrink. concern with gel validation is that it is not very sensitive Several free software programs are available to analyze and therefore may be inconclusive. USA) is a 24 . Experimental design Allele variants are two or more unique forms of a gene that occupy the same chromosomal locus. which is considered the best method The AutoDimer program (authored by P. annealing. while bioinformatics competition for reaction components can contribute to a analysis of primer sequences can greatly minimize the reaction efficiency outside the desirable range of 90-110%. and the same volume from each mixture was loaded on an agarose gel. Prior to the thermal cycling reaction. it is still necessary to monitor The last major concern. The positive side of this potential problem is that primer-dimers are usually Figure 16. impacting The traditional method of screening for primer-dimers reaction sensitivity. dissociation curve. or a single primer folding upon itself. and self-folding in primers and probes Figure 15. Allele variants should be considered when designing primers. Agarose gel analysis to investigate primer-dimer forma- a less favorable interaction than the intended primer. The main concern with primer-dimers is that they may cause false-positive results. Typically. dimerization. This is of particular concern bioinformatics tool that can analyze a full list of primers at with reactions that use DNA-binding dyes such as the same time (Figure 15). GC content differences between variants may alter amplification efficiencies and generate separate peaks on a melt curve. some extension can occur. However.M. risk of dimer formation. in which they appear as diffuse. National Institute of Standards and Technology. efficiency and dynamic range may still be affected. Primer- dimers are of greater concern in more complex reactions such as multiplex real-time PCR. Alternately spliced variants should also be considered when designing primers. gel analysis your real-time PCR primer designs and determine if is useful for validating data obtained from a melting/ they will be prone to dimerize or fold upon themselves. Transcripts originating from these variants can vary by one or more mutations. A screen capture from AutoDimer software. the nucleic acid sample was template interaction. Vallone. resulting in products that approach the size of the intended amplicon and become more abundant as cycling progresses.

target sequences using algorithms that incorporate the primer-dimer formation is not favored. and Vector NTI® Software. there are some cases in which primer-dimers are present in the reaction. The melting curve peak obtained of primer specificity. Smaller. which denatures amplicons and has limited sensitivity. Note that primer but may not affect the overall accuracy of the real-time design software programs. A sharp decrease and sensitivity. for the no-template control (NTC) can be compared to While nonspecific amplification should always be the peak obtained from the target to determine whether eliminated. the presence of these secondary products is not always Ideally. When template is present. melting temperature than that of the desired amplicon and also appearing in the NTC reactions are quite often dimers. melt curve analysis provides the most in fluorescence will be observed at the Tm for each product confidence in confirming gel electrophoretic assessment generated during the PCR. a band may still mask similar-sized fluorescence is high. gel runs of product can often validate the size of the The following recommendations are offered for designing product corresponding to the melting peak. While it can dyes for detection. a single distinct peak should be observed for each a major concern. lifetechnologies. primers are much more likely can automatically design primers for specific genes or to interact with each other.com 25 . the instrument ramps from help to identify products that differ in size from your low temperature. if alternate isoforms or reaction containing template. Amplification of nonspecific products is mismatch stabilization of concern because they can contribute to fluorescence. Designer. such as our web-based PCR assay. primers for real-time PCR : Primer Express®. multiple products are expected. OligoPerfect™ There are situations in which primer-dimers are present. in which DNA is double-stranded and target amplicon. This is not surprising because don’t have to cut-and-paste sequences. • In general. are are present in the NTC but do not appear in reactions seamlessly connected to our online ordering system. so you containing template DNA. resulting in a decreased dynamic primers employed in an assay and within each primer range and decreased data accuracy. • Choose primers that have compatible Tm values which in turn artificially shifts the Ct of the reaction. broader peaks at a lower targeted. and no peaks should be multiple alleles that differ in GC content are knowingly present in the NTCs. Experimental design Melting or dissociation curves should be generated Standard gel electrophoresis is generally the first step following any real-time PCR run that uses DNA-binding in any analysis of real-time PCR specificity. primer-dimers are not an issue. curve. In brief. As long as the peak following guidelines and can also perform genome-wide seen in the NTC is absent in the plus-template dissociation BLAST® searches for known sequence homologies. A common observation is that primer-dimers OligoPerfect™ Designer and Vector NTI® Software. These programs in the absence of template. which helps to prevent imperfect match. Due to its accuracy DNA and results in lower fluorescence. if possible. Nonspecific products are an even greater concern in absolute quantification assays in which precise copy numbers are reported. to high temperature. They (within 1°C of each other) can influence reaction efficiency through competition for • Avoid sequence complementarity between all reaction components. Primer design considerations 2 Again. For example. design primers that are 18–28 nucleotides Primer-dimers are part of a broad category of nonspecific in length PCR products that includes amplicons created when • Avoid stretches of repeated nucleotides a primer anneals to an unexpected location with an • Aim for 50% GC content.

extractions and does not require phenol. including degraded RNA is eluted in water. and organic solvents. Bioanalyzer ® system trace) as the 18S rRNA. consider the source material (cells or tissue) and potential technique limitations. the 28S techniques. which in general is better than assessing the This method is even less time-consuming than organic rRNA peaks alone. Typically. or alcohol can rRNA is twice as bright (or has twice the area under the peak in the also dramatically reduce cDNA synthesis efficiency. and purity of silica-binding methods. The RNA yields Researchers are then able to compare RIN values for RNA may not be quite as high. samples are lysed and homogenized in the presence of guanidine isothiocyanate. Guanidine and ethanol carryover due to incomplete washing can still occur and would have the same deleterious effects on cDNA synthesis efficiency. chloroform then is added and the mixture is separated into aqueous and organic phases by centrifugation. one must speed. RNA remains exclusively in the aqueous phase in the presence of guanidine isothiocyanate. and resulting In assessing RNA quality and quantity. With most silica bead or filter–based methods. Agilent Bioanalyzer ® system trace and gel image display- may result in higher DNA carryover compared to other ing RNA integrity. need for organic solvents. salt guanidine isothiocyanate and phenol itself. Residual guanidine. Guanidine isothiocyanate is a chaotropic salt that protects RNA from endogenous RNases (Biochemistry 18:5294 (1979)). The RNA is then recovered from the aqueous phase by precipitation with isopropyl alcohol. DNA and RNA isolation techniques Assessing RNA quality vary in ease of use. Many protocols use a phenol and Bioanalyzer® system (Figure 17). between 1. polysaccharides. lipids. phenol. though A260/A230 ratio is helpful in evaluating the carryover of most of the same guidelines also hold true for DNA components containing phenol rings such as the chaotropic isolation. Assess RNA integrity on 2 effective method for purifying RNA from a wide variety of a denaturing gel or on an instrument such as the Agilent cell and tissue types. which can lower reaction efficiency. Ensure that the A260/A280 ratio is case of RNA isolation). there are a few nucleic acid purity with regards to carryover of DNA (in the key points to focus on. Prior to performing nucleic acid purification. In general. products. guanidine isothiocyanate mixture to disrupt cells and dissolve cell components while maintaining the integrity of the nucleic acids by protecting them from RNases. but requires the use of toxic chemicals and Figure 17.8 can indicate protein contamination. 26 . and RNA is bound to silica-based beads or determination one step further with the assignment filters and impurities are effectively removed by washing of a RIN (RNA integrity number) value. Intact mammalian total RNA shows two bands or peaks representing the 18S and 28S rRNA species. The purified total calculated from the overall trace. ethanol is added to The Agilent Bioanalyzer® system takes RNA quality the sample. which are One-step reagent-based organic extraction is a very inhibitory to enzymatic reactions.0. and purification and maintenance of consistency.8 and 2. protein. while DNA and protein are driven into the organic phase and interphase. This process is relatively fast and can yield high levels   of RNA. but the purity with regards to from different tissue types to assess quality standardization protein. Experimental design 2. DNA. The This section will primarily discuss RNA isolation. After homogenization. reagents is generally better.4 Nucleic acid purification and quantitation Real-time PCR nucleic acid purification Lastly. A ratio below 1. The RIN value is (Proc Natl Acad Sci USA 76:615 (1979)). methods combining organic lysis with silica columns methods can offer the benefits of good sample lysis with the ease.

in and removes divalent cations from the reaction buffer. RNA. 2 consume a considerable amount of the sample during the unlike in-solution treatments. It can remove even trace measurements cannot distinguish between RNA and DNA quantities of DNA. In addition to removing because Quant-iT™ Assay Kits only report the concentration DNase from reactions. contaminants commonly DNase treatment can occur either in solution or on column. present in samples of purified nucleic acid contribute to UV depending on the isolation method. because salt washes dyes available. fluorescent dyes such as studies RiboGreen® and PicoGreen® dyes are superior to UV Previously. The Qubit® Quantitation heat-inactivation of the DNase at 65°C. the inactivation reagent also binds of the molecule of interest (not contaminants). Platform uses Quant-iT™ fluorescence technology. Finally. which is catalytically 260 nm than do nucleic acids. UV absorbance superior to wild type DNase  I. which can plague RT-PCR reactions. we nucleotides. most UV absorbance readers treatments are common with silica matrix extraction. Experimental design Quantitation accuracy Genomic DNA carryover in expression For quantitation of RNA. This specificity enables DNA-free™ kits help circumvent these problems by using a more accurate results than with UV absorbance readings. and. and high. in the same sample. we described how primer design was the first absorbance measurements because they are designed step toward eliminating DNA amplification in a real-time to have higher sensitivity. lifetechnologies. it is possible to find reagents that overcome remove the enzyme itself. RT-PCR reaction. The drawback is that on-column all of these limitations: dyes that can distinguish nucleic reactions require much more enzyme. In addition. UV absorbance measurements isolation stage is a method by which DNA can be controlled cannot distinguish between nucleic acids and free at the source. or protein. with required for the reaction. With the wide variety of fluorescent inactivated in the presence of EDTA. acids from free nucleotides. In fact. higher accuracy. free nucleotides absorb more at offer super-active TURBO™ DNase. can cause magnesium-dependent advanced fluorophores that become fluorescent upon RNA hydrolysis at this temperature. And. novel DNase inactivation reagent. DNA-free™ and TURBO binding to DNA. Similarly. Free magnesium. and dyes that are insensitive In-solution DNase reactions have traditionally required to common sample contaminants. DNase treatment of the sample at the RNA throughput capability. RT-PCR reactions where they can affect reaction efficiency. quantitation methods using fluorescent dyes are alleviates concerns about introducing divalent cations into very sensitive and only require small amounts of sample. This general. On-column DNase absorbance readings. In addition to traditional DNase I enzyme. dyes that can distinguish DNA from RNA in the same sample. they do not need to be heat- measurement itself.com 27 .

and assay design. only 10% of the cDNA synthesis buffers are a compromise solution that provide acceptable reaction is used in real-time PCR. Once RNA-DNA heteroduplexes for the next stage of complete. The specific this single-tube procedure. the RT is favored by a buffer • RT enzymes and buffers can inhibit real-time that is not optimal for the DNA polymerase. One-step and two-step qRT-PCR making it optimal for rare or limited samples The choice between one-step and two-step qRT-PCR • Sensitivity—two-step reactions may be more comes down to convenience. activity can drastically reduce the yield of full-length The benefits of two-step qRT-PCR include: cDNA. Native RTs perform ideally between conditions. all cDNA produced is amplified level of inhibition will depend on the RT. the reverse transcription is performed and functions in vivo to cleave the RNA strand of in a buffer optimized for the reverse transcriptase. However. secondary start in one-step reactions. the relative in the PCR stage. Thus. An ideal reverse transcriptase will exhibit the following attributes: • Increased risk of primer-dimer formation—forward and reverse gene-specific primers. Experimental design 2. • Less convenient—two-step reactions require more • Contamination prevention—the closed-tube system handling and are less amenable to high-throughput prevents introduction of contaminants between the applications RT and PCR stages • Contamination risk—increased risk of contamination • Convenience—the number of pipetting steps is due to the use of separate tubes for each step reduced and hands-on time is minimized 28 . Normally. manipulations of PCR amplification these native enzymes have resulted in variants with ideal The drawbacks of one-step qRT-PCR include: properties for qRT-PCR. one-step PCR— typically. This can be especially problematic in 42°C and 50°C. first-strand cDNA created is available for real-time but produces lower yields. abundance of the target. sensitivity. the reverse transcriptase and you can interrogate multiple targets from a single thermostable DNA polymerase are both present during RNA sample reverse transcription. sensitive than one-step reactions because the RT The advantages and disadvantages of each technique must and real-time PCR reactions are performed in their be evaluated for each experiment. have a greater tendency structure can have a major impact on the sensitivity to dimerize at the 42–50°C reverse transcription of a reaction. Several RTs. approximately 10% of the cDNA is transferred replication. whereas thermostable RTs function 2 reactions that use DNA-binding dyes for detection at the higher end of (or above) this range and allow • cDNA is not available for other real-time PCR for successful reverse transcription of GC-rich reactions—one-step reactions use all the cDNA from regions.5 Reverse transcription considerations Reverse transcriptases • High-throughput sample screening—for the reasons Most reverse transcriptases employed in qRT-PCR are mentioned above derived from avian myeloblastosis virus (AMV) or Moloney • Sensitivity—one-step reactions may be more murine leukemia virus (M-MLV). This slightly and associated buffer components may inhibit the lower functionality is compensated by the fact that. time PCR reactions—two-step qRT-PCR produces enough cDNA for multiple real-time PCR reactions. the sample is lost • Reduced RNase H activity—The RNase H domain is present in common native reverse transcriptases In two-step qRT-PCR. Native AMV reverse sensitive than two-step reactions because all the transcriptase is generally more thermostable than M-MLV. RNase H into each real-time PCR reaction. most notably SuperScript® II and III. For qRT-PCR applications. which translates to poor sensitivity. so if the reaction fails. and the robustness of the The benefits of one-step qRT-PCR include the following: amplification reaction. using DNA polymerase if not diluted properly. have been • cDNA may be archived and used for additional real- engineered for reduced RNase H activity. also in its optimal buffer. because the RT but not optimal functionality of both enzymes. present from the • Thermostability—As discussed earlier. individually optimized buffers • Multiple targets—depending on the RT primers used. and the RT is inactivated in the high-temperature DNA polymerase activation stage (the The drawbacks of two-step qRT-PCR include: so-called hot start). the RT step. In a one-step reaction.

More thermostable RTs may RT efficiency and consistency and. In addition. data perform better with longer primers. degradation may prevent the RT from creating cDNA that corresponds with full-length target amplicon. produce a cDNA copy of the target gene product. Efficiency variations that are not normalized can result in inaccurate conclusions. individual targets may respond Oligo(dT) primers are a favorite choice for two-step differently to one primer choice over another. paraffin-embedded (FFPE) samples. such as that isolated from efficiency formalin-fixed. Depending on the first-strand primer used. can use gene-specific. However. as long as the real-time than the PCR stage. they do not offer the structure do not pose as much of a problem as they do with flexibility of oligo(dT) and random primers. Because they anneal throughout and have been shown to be the most consistent of the the target molecule. and RT enzyme employed: RNA expression level comparisons are more accurate if the RT is less sensitive to the inevitable Figure 18. Employing qRT-PCR reactions always employ a gene-specific primer a combination of random and oligo(dT) primers can for first-strand synthesis.com 29 . and therefore can offer the highest sensitivity in real- time PCR. generally an important concern. may The RT stage of a qRT-PCR reaction is less consistent also be problematic. or random primers Choosing the best oligo(dT) primer may depend in part (Figure 18). degraded transcripts and secondary primer options for RT. and primer selection can play a large role in on the reaction temperature. Experimental design RNA priming strategies oligo(dT)12–18 is a mixture of 12-mer to 18-mer thymidines. each reactions because of their specificity for mRNA and primer option should be evaluated during the initial assay because many different targets can be analyzed from the validation stage to determine which provides optimal same cDNA pool when they are used to prime reactions. cDNA. RNA sample complexity. factors associated with the starting sample. Graphical representation of commonly used RNA-priming strategies. The first-strand synthesis reaction slippage by annealing at the 3�  UTR/poly(A) junction. which remain more accuracy. Because of this. in that they only 2 gene-specific primers and oligo(dT) primers. consequently. Sequence-specific primers offer the greatest specificity such as bacterial RNA. sensitivity and accuracy. Reverse transcription is typically the most variable portion Anchored oligo(dT) primers are designed to avoid poly(A) of a qRT-PCR reaction. because they always initiate reverse transcription at the 3� end of the transcript. Nonetheless. data has shown that for studies involving scarce or precious samples. Oligo(dT)20 Differences in RNA integrity: The degradation level of is a homogeneous mixture of 20-mer thymidines. gene-specific primers are typically not the best choice While increased yield is a benefit. which the premature termination downstream of this location is not thermostable DNA polymerase isn’t normally tested with. difficult secondary structure Factors influencing reverse transcription may lead to incomplete cDNA generation. Ideally. the less sensitive the PCR assay will be. However. This is due to a combination of PCR primers are designed near the 3� end of the target. One-step random primers can overestimate copy number. of both in the same RT reaction. These factors include: Multiple types of oligo(dT) primers are available. while a particular RNA sample has a direct impact on the percentage of mRNA target that is converted into cDNA and therefore quantified. lifetechnologies. Each primer type presents unique theoretical benefits and drawbacks. whereas other primer options sometimes increase data quality by combining the benefits are compatible with two-step reactions. Oligo(dT) priming of fragmented RNA. GC content. Oligo(dT) primers are not Random primers are great for generating large pools of recommended if 18S rRNA is used for normalization. They are also ideal for non-polyadenylated RNA. Random primers are used only in two-step qRT-PCR reactions. The lower the RT efficiency. tightly annealed at elevated temperatures compared to their shorter counterparts. oligo(dT).

Experimental design

differences between samples. For example, data has can inhibit enzymatic reactions. Variations in the levels
shown that sample complexity alone, meaning all the of these contaminants between RNA samples can affect
background nucleic acid not compatible with the RT primer, sample comparison. Therefore, it is important to use an
can result in as much as a 10-fold difference in reaction RNA isolation method that results in consistently low levels
efficiency. RTs capable of consistent cDNA synthesis in this of these byproducts. We also recommend using a validated
background are ideal. normalizer gene in your real-time PCR reactions.

Carryover of organic solvents and chaotropic salts:
Ethanol and guanidine are necessary for RNA capture but

2.6 Controls
Controls in real-time PCR reactions prove that signals type of contamination can make expression levels look
obtained from experimental samples represent the higher than they actually are.
amplicon of interest, thereby validating specificity. All
No-RT reactions should contain all reaction components
experiments should include a no-template control (NTC),

2
except the reverse transcriptase. If amplification products
and qRT-PCR reactions should also include a no-reverse
are seen in no-RT control reactions, it indicates that DNA
transcriptase (no-RT) control.
was amplified rather than cDNA. This can also artificially
NTC controls should contain all reaction components inflate apparent expression levels in experimental
except the DNA or cDNA sample. Amplification detected samples.
in these wells is due to either primer-dimers or
contamination with completed PCR reaction product. This

2.7 Normalization methods
Earlier in this guide, we indicated that eliminating in efficiency in reverse transcription and real-time PCR
experimental inconsistencies should be a paramount reactions. For example, minute differences in the levels
concern for real-time PCR experimental design. Deviating of contaminants can affect reverse transcription reactions
from the experimental plan can limit researchers’ ability and lower amplification efficiency. Any variation in samples
to compare data and could lead to erroneous conclusions is then amplified during PCR, with the potential to result
if deviations are not accounted for in the analysis. Sources in drastic fold-changes unrelated to biological conditions
of experimental variability include the nature and amount within samples. Pipetting is also subject to operator
of starting sample, the RNA isolation process, reverse variation, and there is no normalization to compensate for
transcription, and, of course, real-time PCR amplification. it in post-purification RNA analysis.
Normalization is essentially the process of neutralizing the
Normalizing to a reference gene: The use of a normalizer
effects of variability from these sources. While there are
gene (also called a reference gene or endogenous control)
individual normalization strategies at each stage of real-
is the most thorough method of addressing almost every
time PCR, some are more effective than others. These
source of variability in real-time PCR. However, for this
strategies include:
method to work, the gene must be present at a consistent
Normalizing to sample quantity: Initiating the RNA or DNA level in all samples being compared. An effective
isolation with a similar amount of sample (e.g., tissue or normalizer gene controls for RNA quality and quantity,
cells) can minimize variability, but it is only approximate and differences in both reverse transcription and real-time
and does not address biases in RNA isolation. PCR amplification efficiencies. If the RT transcribes or the
DNA polymerase amplifies a target at different rates in two
Normalizing to RNA or DNA quantity: Precise quantification
different samples, the normalizer transcript will reflect
and quality assessment of the RNA or DNA samples
that variability. Endogenous reference genes, such as a
are necessary, but fall short as the only methods for
“housekeeping” gene, or exogenous nucleic acid targets
normalization, because they do not control for differences
can be used.

30

Experimental design

Endogenous controls
Common endogenous normalizers in real-time PCR
include:

• β-actin (ACTB): cytoskeletal gene
• 18S ribosomal RNA (rRNA): ribosomal subunit
• Cyclophilin A (PPIA): serine-threonine phosphatase
inhibitor
• Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH): glycolysis pathway
• β-2-microglobulin (B2M): major histocompatibility
complex
• β-glucuronidase (GUSB): exoglycosidase in lysosomes
• Hypoxanthine ribosyltransferase (HPRT1): purine Figure 19. Gene expression levels of commonly used endogenous
salvage pathway controls and the importance of normalization. In this example, two
treatment groups and a normal group were analyzed for the expression
• TATA-Box binding protein (TBP): RNA transcription levels of common reference genes, which were amplified in addition
Because every real-time PCR experiment is different,

2
to an internal positive control (IPC). The IPC provides a standard for
thought and careful planning should go into selecting a normal reaction-to-reaction variability. The bars represent up- or down-
regulation of the normalizer in each treatment group as compared to the
normalizer. Instead of choosing a normalizer based on normal sample, which is represented by a ΔCt of 0. The goal is to find a
what others in the lab use, choose one that best supports normalizer that mimics the changes exhibited by the IPC.
the quantification strategy of the specific target.

The first requirement of a quality normalizer is that it is • GAPDH is a common normalizer that has been shown
similar in abundance to your target gene product. This to be consistent in many cases. However, GAPDH is
is especially important when multiplexing, because upregulated in some cancerous cells, in cells treated
if the normalizer reaction plateaus before the target with tumor suppressors, under hypoxic conditions,
Ct is reached, the normalizer itself will impede target and in manganese or insulin-treated samples.
amplification, causing a higher target Ct, thus defeating • β-actin is another commonly employed housekeeping
its purpose. Alternatively, the normalizer reaction can gene because it exhibits moderately abundant
be assembled with primer-limited conditions to mimic a expression in most cell types. However, its
lower expression level. Most TaqMan® Endogenous Control consistency has been questioned in breast epithelial
Assays are available with primer-limited configuration. cells, blastomeres, porcine tissues, and canine
Real-time PCR assays for normalizer targets should have a myocardium.
similar amplification efficiency to assays for experimental • 18S rRNA constitutes 85–90% of total cellular RNA
targets; this can be evaluated using a standard curve. and has been shown to be quite consistent in rat
Although correction factors can be applied when comparing liver, human skin fibroblasts, and human and mouse
reactions with different efficiencies, accuracy is enhanced malignant cell lines. However, its level of abundance
when the reaction efficiencies are close to one another. makes it a problematic normalizer for medium- and
low-expressing targets. Often it is difficult to find a
Last and most important, expression of the normalizer concentration of RNA at which 18S rRNA provides
should be consistent, regardless of the treatment or a wide enough baseline and also at which the
disease state of the sample. This must be experimentally target of interest generates a Ct within 40 cycles. In
determined, as shown in Figure 19. addition, multiplexing may necessitate limiting the
While multiple replicates should be performed to ensure concentration of 18S primers so that the normalizer
accuracy, it is clear that cyclophilin A (PPIA), TBP, and HPRT1 doesn’t sequester all the reaction components and
would not be good normalizer choices for these treatment make PCR conditions unfavorable for the target of
groups because they appear to be down-regulated with interest.
treatment. The expression levels of even the most common
Alternative methods exist that do not rely on the accuracy
reference genes can be altered under certain conditions
of a single reference gene, but rather the geometric mean
and therefore should always be validated:
of multiple validated normalizers. This use of multiple

lifetechnologies.com 31

Experimental design

consistent normalizers may prove to be a better buffer An example of an exogenous normalizer is an in vitro-
against the Ct fluctuations of any single gene, thereby transcribed RNA specific to plant processes, such as a
increasing assay and sample type flexibility. photosynthetic gene. This could be spiked into mammalian
samples because those cells would not have this same
Exogenous normalizers transcript endogenously.
Exogenous normalizers are not as commonly employed, but The drawbacks to employing an exogenous normalizer are:
are a viable alternative if a highly consistent endogenous
normalizer cannot be found for a specific sample set. • It is not endogenous. Maximize the utility of
An exogenous reference gene is a synthetic or in vitro exogenous normalizers by spiking them into the
transcribed RNA with a sequence that is not present in the workflow early, for example into the cell lysis buffer.
experimental samples. Due to its exogenous origin, it does • Accuracy is subject to pipetting variability when
not undergo the normal biological fluctuations that can introducing the normalizer.
occur in a cell under different conditions or treatments. • Transcript stability may be affected by prolonged
When using exogenous normalizers, the earlier they are storage and multiple freeze-thaws. Therefore, copy
added to the experimental workflow, the more steps they number should be routinely assessed to ensure it has
can control. For example, if an exogenous transcript is not shifted over time.

2
added to the cell lysis buffer, it can be used as a normalizer
for cell lysis, RNA purification, and subsequent RT and
PCR reactions.

2.8 Using a standard curve to assess efficiency,
sensitivity, and reproducibility
The final stage of experimental design is validating that curve slopes of –3.6 to –3.1. The graph in Figure 20 shows
the parameters discussed up to this point result in a highly the measurement bias resulting solely from differences in
efficient, sensitive, and reproducible experiment. reaction efficiency.

Validating the reaction efficiency for all targets being
Reaction efficiency compared (e.g., reference genes and genes of interest),
As discussed previously, the overall efficiency of a real- optimizing those efficiencies to be as similar as possible,
time PCR reaction depends on the individual efficiencies of and employing efficiency corrections during data analysis
the RT reaction and the PCR amplification reaction. can reduce these effects. These strategies will be discussed
RT efficiency is determined by the percentage of target further in the data analysis section.
RNA that is converted into cDNA. Low conversion rates can Reaction efficiency is best assessed through the generation
affect sensitivity, but variation in the conversion percentage of a standard curve. A standard curve is generated by
across samples is of greater concern. creating a dilution series of sample nucleic acid and
PCR amplification efficiency is the most consistent factor performing real-time PCR. Then, results are plotted
in a real-time PCR reaction. However, this amplification with input nucleic acid quantity on the x-axis and Ct on
exponentially magnifies slight variations in RT efficiency, the y-axis. Samples used to generate the standard curve
potentially resulting in misleading data. 100% efficiency should match (as closely as possible) those that will be
corresponds to a perfect doubling of template at every used for the experiment (i.e., the same total RNA or DNA
cycle, but the acceptable range is 90–110% for assay sample). The dilution range, or dynamic range analyzed for
validation. This efficiency range corresponds to standard the standard curve, should span the concentration range

32

standard curves for three different targets quantities that may be measured in the real-time PCR were generated. To some. true gauge of sensitivity of an assay is whether a given An example of this type of comparison would be when a low amount of template fits to the standard curve while normalizer gene is compared against a target gene to adjust maintaining a desirable amplification efficiency. The slope of the Sensitivity and reproducibility curve is used to determine the reaction efficiency. Bias effect caused by different amplification efficiencies.com 33 . However. which is a concentrations and therefore cannot accurately be used for measure of replicate reproducibility. which A standard curve with an efficiency within the desirable most scientists agree should be between 90% and 110%. be repeated over time to assess whether the consistency. purple curve. standard curves can also be used to determine whether the problem with a particular reaction is due to inhibition or lack of optimization. there is a 100-fold difference Figure 21. sensitivity is measured by how early a curves indicates that they have similar efficiencies and target Ct appears in the amplification plot. The parallel nature of the red and blue reaction. the therefore can be accurately compared at any dilution. Experimental design   Figure 20. one with 100% efficiency. is providing an efficiency value for relative quantitation. lifetechnologies. This will be discussed in more detail in the Troubleshooting section. Differences in efficiency become more important in reactions with more cycles and assays that require greater 2 sensitivity. expected for the experimental samples. In addition to assessing the experimental conditions and and therefore the data accuracy for the samples. however. Four different real-time PCR reactions are shown that range from 70% to 100% efficiency. after 30 cycles. maintained. window of 90–110% defines the range of input template In Figure 21. becomes less efficient at the lower The standard curve also includes an R2 value. The most for non-biological variability from sample to sample. The divergence is not necessarily apparent in the early cycles. The dilute sample that fits determines reaction sensitivity. Standard curves may comparison purposes at these lower concentrations. Example standard curve used to evaluate the efficiency of in reported copy number between a reaction with 70% efficiency and real-time PCR. However.

Plate preparation 3 .

3 Plate sealing 36 3.2 Plate loading 36 3.1 Mixing 36 3. Plate preparation 3.4 Plate insertion 37 3 lifetechnologies.com 35 .

if you are analyzing the expression of 20 with the assay master mix. such as unintended one side. because nucleic acids are hydrophilic and will quickly mix On the other hand. 3. 1-2 seconds are highly efficient mixing methods. Use it to smooth down the cover. plastic installing tool is provided with the covers. which are thin sheets of plastic with adhesive on bottom. Since real-time PCR master mixes are typically efficiency. plan your are analyzing the expression of 5 genes in 20 different RNA pipetting to avoid cross-contamination of samples and samples. Also. as the ink could be pull up the cover. The adhesive is protected by a white backing. into the reaction plate or tube first and then add sample. so as not to do not write on the bottom of plates. Regardless of the order of reagent addition. the cover is in good contact with the plate. Note any perforations.3 Plate sealing Plastic PCR plates may be sealed with optical caps or optical Once the plate has been sealed. because. Centrifuge touching the cover itself. and air bubbles at the bottom of the well. 36 . precision could be compromised. white strips are removed by the perforation. Use these tabs to handle the cover without empty wells or wells with abnormal volumes. A square. substances can adversely affect real-time PCR data. then centrifuge all real-time Gently inverting tubes a few times or lightly vortexing for PCR reaction components just before assembling reactions. For example. Mixing fully assembled reactions is not necessary. as this will may also be used to hold down each end of the cover as the interefere with fluorescence excitation and reading. Plate preparation 3. Remove the white backing and the plate briefly to correct any adherent drop and bottom- place the cover on top of the plate.2 Plate loading Base the order of reagent addition into reaction plates or to first dispense the RNA samples. Always centrifuge briefly to collect the contents denser than the other real-time PCR reaction components. On empty wells. and thawed nucleic acid reduce enzyme activity that could reduce amplification samples. especially along the 4 plates are available with bar codes. from the solutions. if you mix. then add assay master tubes on the nature of the experiment. Gently swirl enzyme-containing master mixes and briefly avoid over-vortexing because it can cause bubbles to form (1–2 seconds) vortex other components such as PCR that could interfere with fluorescence detection. Alternatively.1 Mixing It is a good idea to briefly mix. hold it up and inspect the covers. Inspect the top of the plate to verify that transferred to the block in the real-time PCR instrument. However. at the bottom of the container and eliminate any air bubbles it is important to adequately mix reaction mixtures. genes in only 5 different RNA samples. Plates may be safely labeled along the skirt. looking for any anomalies. drops of liquid adhering to the walls of the the ends of the cover are rectangular tabs delineated with well. otherwise. it would be easier 3 3. especially along Significant block contamination by colored or fluorescent the 4 upper edges. and can primer pairs or TaqMan® Assays. inside the raised edges. Do not write on edges at the top of the plate. The edge of the installing tool the surface of the plate over well positions. bubble problems. it would make more sense to dispense assay mix assays.

it may be loaded into the real-time Fast block are not the same. If pulled out. even if Fast They can be routinely stored at room temperature under mode is not being used. normal laboratory lighting for days without ill effect. Avoid direct exposure of loaded plates to sunlight. Note that a 96-well standard block and 96-well 3 lifetechnologies. whereas Fast Plates containing DNA or cDNA template and AmpliTaq 96-well plates and Fast tubes can accommodate 0. be sure that the block matching the plate type is installed.com 37 . which is computer controlled. For some instruments. plate is placed on an arm. For instruments with interchangeable thermal cycling blocks. the aluminum foil to protect it from sunlight. wrap the plate in the drawer is pushed back in. Plate preparation 3. Fast plastics must be used with a Fast block. this Some instruments have a drawer system: the drawer is can compromise the fluorescent dyes in the mixture. Gold® reagents are very stable at ambient temperature. The loading process varies with the model of instrument. following the manufacturer’s instructions.2 mL capacity. Standard 96-well plates PCR instrument.1 mL. the plate is placed in the block or holder and you need to transport a plate outside.4 Plate insertion Once the plate is ready. and standard tubes have a 0.

Data analysis 4 .

1 Introduction 40 4. Data analysis 4.4 High resolution melting (HRM) curve analysis 44 4.5 Multiplex real-time PCR analysis 46 4 lifetechnologies.3 Comparative quantification 42 4.com 39 .2 Absolute quantification 40 4.

standard curve to determine their copy number. the template used for absolute standard the target of interest is accurately determined to contain curve generation will determine the accuracy of the data. The following types of template have been used as absolute quantification standards: 1. selecting the right quantification method depends planning. The template for standard curve generation as well as magnitude. the copy number of the 4 target of interest must be known.1 Introduction As mentioned in the beginning of the experimental design • Comparative quantification still requires careful section. Workflow for standard curve setup for absolute quantification. 2 x 1011 copies. a target template solution of known concentration is diluted over several orders of 1. To perform In absolute quantification. This is the method of choice for gene expression studies and • Absolute quantification determines actual copy offers two main options for quantification: ΔΔCt and numbers of target. and real-time PCR is performed on for initial copy number determination. pure template down to 2 x 103 copies. Absolute quantitation is often used for determining viral titer. the template is accurately quantified. 2. consider the following: absolute quantification. particular Ct. or plasmid clone containing the target of interest. prior Template choice for standard curve to curve generation. but the data generated are for relative on the goals of the experiment. and the data are the method used to quantify that template is the foun- used to generate a standard curve in which each target dation for the experiment. copy number of the target under investigation. but is also the most labor- standard curve quantification. using at least three replicates. intensive and difficult form of quantitation. 40 . Although you may need homogeneous. A sample of As mentioned. amplified by real-time PCR. dilution series is essential. DNA standards: PCR amplicon of the target of interest. With an absolute standard curve. quantify. The sample is diluted 10-fold eight times. for generation of each dilution. Also remember that real- The unknown sample Ct values are then compared to this time PCR sensitivity amplifies minute human error. This dictates that. 4. generation Figure 22 highlights a standard curve setup.2 Absolute quantification Absolute quantification is the real-time PCR analysis of curve. and maintain stability with proper storage Cons: Cannot undergo the reverse transcription step of qRT-PCR. Similar reverse transcription and PCR efficiencies for the target template and dilution series of the actual Standard curve generation—overview samples are critical. Pipetting accuracy for the concentration is plotted against the resulting Ct value. The accuracy of the quantification is directly related choice for researchers who need to determine the actual to the quality of the standard curve. Pros: Easy to generate. This method requires thoughtful planning and a highly accurate standard curve. The resulting the standard curve it is best to use target template that standard curve correlates each copy number with a is as similar to the experimental samples as possible. which can impact reaction efficiency significantly Figure 22. this includes subjecting it to most of the same processing steps as the experimental samples. abundance rather than exact copy number. Data analysis 4. The copy number values for the unknown Because steps such as nucleic acid isolation and reverse samples are then derived by comparison to this standard transcription play a role in reaction dynamics.

Figure 24. The high and low Ct values are discarded. The PCR product generated from the real-time PCR itself can be reamplified with a 5� T7 promoter-containing sequence and a 3� poly(T)-containing reverse primer.4. unrelated yeast tRNA can be added at a 1:100 cRNA to tRNA ratio to mimic the Cons: Time-consuming to generate and difficult to 4 normal background of biological samples. 2. It is important that the dilution series suppress the cDNA synthesis rate as much as 10-fold. When calculating the average threshold cycle (Ct) value. Data analysis Figure 23. such as yeast tRNA. Another precise method of quantification is digital PCR. For example. lifetechnologies. Standard curve application—cRNA the lowest point in the standard curve should not contain To demonstrate how the recommendations for an absolute 100 template copies if it is possible that an unknown test quantification standard curve should be applied. this sample may contain only 10 copies. and each dilution within each series is amplified efficiency. can be spiked minimize this effect. we can see how the Ct values plasmid or a PCR product. Schematic diagram of the in vitro transcription protocol. the highest and lowest values obtained from replicate reactions can be discarded. standards means that they will often exhibit higher Pipetting inaccuracies can have a significant effect on efficiencies than experimental samples. This standard is maintain accuracy over time due to instability then diluted over at least 5 to 6 orders of magnitude for use The homogeneous nature of each of the RNA and DNA in Ct determination by real-time PCR. absolute quantification data. Pros: Incorporates RT efficiency and mimics the target of interest most similarly With the copy number determined. Therefore. encompass all possible template quantities that may be encountered in the experimental samples. three separate cRNA dilution series are heterogeneous environment and help to balance reaction prepared.com 41 . six Ct values will be obtained. which corresponds to accuracy. for a given sample vary by as many as 2 cycles. RNA standards: In vitro–transcribed RNA of the target RNA (cRNA) is recommended over UV absorbance of interest (Figure 23). measurement. the average Ct value of 21. It has been shown that background RNA can in duplicate. This is Because it has an extended limit of detection and better minimized by assigning this dilution. section will walk step by step through the creation of a cRNA standard curve for this method of quantification. and the remaining four T7 RNA polymerase can be used to generate a Ct values are averaged. fluorometric measurement of complementary a particular copy number. Appropriate precautions can background RNA. As can be seen in the plate setup into the standard template to create a more realistic in Figure 24. For each dilution. The in vitro transcription reaction produces polyadenylated sense mRNA. it will be accurately quantified and diluted for the standard curve. If we focus on the 10-4 dilution in homogeneous pool of the transcript of interest from a this example (Figure 24). After purification. Plate setup for standard curve generation.

but it does duplicate. this small deviation in efficiencies still opens the door to inaccuracies.. 42 . The fold difference is then simply The requirement for the ΔΔCt method is that the efficiencies 2 to the power of ΔCt. untreated or wild type sample) and a normalizer calibrator (normal) sample and one or more experimental (e. avoid the assumptions made with previous techniques. effects of experimental variability on this result is unknown. Comparative quantification algorithms— standard curve method The standard curve method of comparative quantification employs the Ct difference between the target gene in the test and calibrator samples. Data analysis 4. which instead focuses on and calibrator sample are now adjusted in relation to a fold change compared to the calibrator sample. Fold difference = 2-ΔΔCt Comparative quantification algorithms— ΔCt sample . Of course.g. average ΔCt between the normalizer and target gene can be obtained for each dilution. However. In this technique. is not quite as rigorous as absolute ΔΔCt quantification. normalizer (norm) gene Ct from the same two samples. which applies to most The ΔΔCt method is a very popular technique that compares gene expression studies. Here we outline the common methods of comparative The resulting ΔΔCt value is incorporated to determine the quantification and how variability is controlled in each. the calculated relative expression level requirement of this technique is that the normalizer gene of the target gene in the treated sample is 64-fold lower than that of the calibrator. while still technically Comparative quantification algorithms— challenging. for both the normalizer and target gene are identical. sample quality.3 Comparative quantification Comparative quantification. Precise copy number determination is not for the gene of interest (GOI) in both the test sample(s) necessary with this technique. Ct values samples. the calibrator Ct was 6 and it was 12 for the treated sample. housekeeping gene). the expression level of a gene results from experimental samples with both a calibrator of interest is assayed for up. and the difference between them is the ΔCt. normalized to the reference gene Ct values and adjusted for minute variations in   amplification efficiency.ΔCt calibrator = ΔΔCt ΔCt This is comparative quantification in its most basic form..Ct norms = ΔCt sample A Ct is obtained for expression of the gene of interest Ct GOIc . it is the consistency of that value across each dilution that matters.or down-regulation in a (e. The treated sample and calibrator were run in gene of interest is necessary with this method. 4 To some researchers. Ct GOIs . A standard curve to determine amplification efficiency for both the normalizer and the Figure 25. and thus the conclusion cannot be considered trustworthy. The value itself is not important.g. A According to the ΔCt method.Ct normc = ΔCt calibrator from both a test and calibrator sample. because a normalizer was not employed. gene of interest using the same samples (Figure 26). The or reaction efficiency (Figure 25). Comparative quantification of expression in a treated sample and calibrator. fold difference in expression. Employing a correction for both the gene of interest and the normalizer minimizes the effects of amplification efficiency variation. With this method. the obvious question is: what range of deviation Fold difference = 2ΔCt is acceptable? The way to determine this is to generate This basic method is inadequate because it does not a standard curve for both the normalizer gene and target control for differences in sample quantity. the be the same across all samples in the analysis.

Relative efficiency plot. lifetechnologies. Efficiencies are derived from both slopes (Figure 27).Ct GOI s ΔCt normalizer = Ct norm c. The ΔCt method does not employ a normalizer. HeLa RNA was With a normalizer employed. a slope of <0.5. While a perfectly flat line (slope = 0) indicates identical efficiency across all input concentrations.0 to 2. Fold difference = (Etarget)ΔCt target /(Enormalizer)ΔCt normalizer E = efficiency from standard curve E = 10[-1 /slope] ΔCt target = Ct GOI c . When plotted against dilution or input amount of RNA.com 43 . In most cases.1 is generally While the same setup for the amplification curves from the considered acceptable when employing the ΔΔCt method. Figure 27. Because copy number is irrelevant. one has the option of fold. diluted over 6 orders of magnitude. Therefore. relative quantification will be the method of choice. The ΔCt ranges from 2. you’ll need to generate a precise standard curve using known quantities of target template.   while the ΔΔCt method involves one or more reference genes to normalize for real-time PCR processing variability. 4 In summary. efficiency values from standard curves (ideally run on the same plate) are now incorporated to adjust the normalizer and gene-of- interest (GOI) Ct values. Keep in mind that any differences between the calibrator sample and the experimental samples may result in an inaccurate efficiency correction   and therefore inaccurate calculations for fold-changes in gene expression. choose the calibrator sample for the standard curve carefully. obtained. a slope is dilutions or values given to those dilutions can be arbitrary. and have similar complexity to the experimental samples. Efficiency values are obtained from standard curves to adjust the normalizer and gene-of-interest C t values.Ct norm s Fold difference equation derived from M.W. total RNA from the cell line or tissue being studied. Pfaffl in A-Z of Quantitative PCR. If you need to know how many target molecules are in the sample. ΔΔCt method are used in this technique. the Figure 26. with or without a reaction-efficiency to generate standard curves for both the normalizer and gene of adjustment. interest (GOI). This calibrator sample should undergo the same purification procedure. Data analysis In Figure 27. the first step in choosing a quantification strategy is to determine whether absolute or relative quantification will best address the questions to be answered. be involved in the same reactions. the perfect calibrator sample is one of the heterogeneous samples containing the target of interest—for example. In order to capture the most accurate efficiency value for the calculation. and real-time PCR was performed change calculations.

sequencing. Mutations in PCR analysis requires dsDNA-binding dyes that are capable of products are detectable by HRM analysis because they distinguishing the melting points of amplicons that differ lead to changes in the shape of DNA melting curves. Rotor-Gene® 6000 shape of the melt curve. closed-tube post-PCR method for identifying SNPs. This technique has opened the door to many new applica- tions for dsDNA-binding dyes. by a single nucleotide. a dramatic decrease in fluorescence is format. or In addition to a specialized instrument and software. 7900HT Fast. After 40 cycles. using decrease due to the fluorescent dye being released. This temperature is known as the Tm. The most widely used HRM analysis application is gene scanning. HRM as an alternative to. HRM analysis applications due to the minute Tm shifts that must be detected. System (Corbett Life Science). of the product. Characteristics of the melt curve profile for a PCR 6/7. Mutations can be detected as either a shift in Tm or a change in StepOne™ Real-Time PCR Systems. a thermo-stabilized form of the • Gene scanning (mutation discovery) fluorescent SYTO® 9 dye with low background fluorescence • Mutation analysis and high brightness in the presence of double-stranded • Single-nucleotide polymorphism (SNP) detection DNA. the amplification products are • Heterozygosity studies annealed and highly fluorescent. and inflexible. and methylation patterns. or melting temperature. until dsDNA-binding dyes. High resolution melting curve profiling requires a real- time PCR instrument with upgraded optical and thermal capabilities as well as analysis software for extremely fast data acquisition and highly accurate thermal control and   consistency. When the HRM analysis • Species identification 4 begins. 44 . In HRM analysis. observed as the sample transitions from double-stranded to single-stranded DNA (Figure 28). discrimination at the single-nucleotide level is one of the more challenging. HRM. instruments currently set up to perform HRM analysis. StepOnePlus™.4 High resolution melting (HRM) curve analysis High resolution melting curve (HRM) analysis is a novel. begin to denature (or melt). Gene scanning is the search for the presence HRM analysis chemistry of unknown variations in PCR amplicons prior to. As the PCR products fluorogenic probe for each target. homogeneous. which was expensive. While many applications exist for HRM. their fluorescence will slowly time-consuming to design. offers the same capabilities as the temperature approaches the PCR product’s Tm. Specific DNA sequences have characteristic melt-curve profiles. and amplicon. In contrast to traditional melt curve analysis. The MeltDoctor™ HRM Master Mix employs MeltDoctor™ HRM Dye. The QuantStudio® 12K Flex. HRM analysis is a more sensitive approach than traditional melt curve profiling in which double- stranded DNA is monitored for the temperature at which it dissociates into single-stranded DNA. and LightCycler® 480 HRM can distinguish between amplicons with just a single-nucleotide System (Roche Diagnostics) are commercially available difference. ViiA™ 7. 7500 Fast. approximately 80–250 bp fragments of genes are amplified using PCR in mixtures that contain a high-performance double-stranded DNA (dsDNA)– Some common applications of HRM analysis include: binding dye. the real-time PCR instrument slowly ramps • Methylation analysis the temperature higher while simultaneously recording These applications traditionally required a unique fluorescence data from the amplicons. novel mutations. Very probe-based analysis in a more inexpensive and flexible close to the Tm. QuantStudio® Figure 28. Data analysis 4. heteroduplex DNA samples show melting curves with different profiles than those derived from homozygous wild type or mutant samples. When amplified and melted.

com 45 . and secondary structure and folding of the PCR 4 product. exon/intron boundaries.) Keys to successful HRM analysis assays • Set an appropriate ramp rate for the instrument. • Ensure specificity of the PCR amplicon. Sufficient pre-and post-melt temperature data are required for accurate curve normalization and high replicate correlation. Data analysis Dyes that have been successfully used for HRM analysis include: • MeltDoctor™ HRM Dye (Thermo Fisher Scientific) • Molecular Probes® SYTO® 9 dye (Thermo Fisher Scientific) • LCGreen® and LCGreen® Plus+ reagents (Idaho Technology) • EvaGreen® dye (Biotium Inc. which is needed for HRM. for some instruments. • Maintain similar fluorescent plateaus.5 mM to 3 mM range. • Avoid amplifying across regions that could contain variations other than those of interest. Starting with similar amounts of template can be helpful. Mispriming products and primer-dimers can complicate data interpretation. and use of a hot-start DNA polymerase will help to obtain high specificity. that offers sensitive and unbiased amplification of the gene fragments of interest. Check for species homology. MgCl2 in the 1. to insert a pre-hold step at 50°C following amplification (but prior to melt) to ensure that all products have reassociated and to encourage heteroduplex formation. lifetechnologies. In general. Assess mispriming using a standard low resolution melt curve. splice sites. Compared to larger amplicons. • It is also recommended. For example. Primer concentrations lower than 200 nM. This will typically result in 10–20 data collection points per degree C. such as MeltDoctor™ HRM Master Mix. Use a mix. the window should have a range of 10°C on either side of the melting temperature of the amplicon (Tm ± 10°C). those around 100 bp will allow easier detection of single-nucleotide melt events. for all targets being analyzed. • Ensure that enough template is used in the reaction. Ct values should be below 30 to generate sufficient material for accurate melt analysis. • Keep amplicons short for highest sensitivity. • Provide a sufficient melt data collection window. and therefore similar PCR product quantities. Differences in quantity between the samples being compared can affect melting temperatures and confound HRM analysis. No-template control (NTC) melt curves are important for evaluating specificity.

including: the primer concentration for those targets that are easier • Primer and probe design to amplify or more abundant can level the playing field. Reducing the combinations) amount of β-actin primers limits its rate of amplification. you should use the lowest primer concentration Primer and probe design that does not delay Ct values. USA). • Higher throughput. such as dNTPs propensity to dimerize in any combination. either reducing the amount of primers or increasing • Less reagent usage. the number of targets that can be amplified interest. reaction with a low-abundance target. However. The following concentrations is most likely to achieve Ct values that are are particularly important factors that will maximize consistent between singleplex and multiplex reactions. other experimental hurdles exist: (authored by P. for higher-abundance targets. One such free program. target abundance. or both.M. and these primers and probes primer designs in a given multiplex reaction for their have to share available PCR components. concentrations. less cost. all primers and probes will be target. More targets can be analyzed per 4 real-time PCR run and per sample. Remember. if β-actin (a high-copy normalizer) is multiplexed concentration. • Validation of the multiplex assay allowing the less-abundant target to be amplified in an unhindered manner. • Perform BLAST® searches with primer and probe designs to ensure their specificity for the targets of Theoretically. those same two targets were amplified in different Ensuring a high efficiency of amplification requires (even adjacent) wells. reaction. Multiple Primer Analyzer. so does the probability that primers and probes will dimerize Reaction component concentrations or that competition for reaction components will limit Every multiplexing reaction is different. Primer concentration and target abundance Keys to a successful multiplex assay Not all amplicons in a multiplex reaction are present in the A successful multiplex reaction must take many factors same numbers nor amplify at the same efficiency. For • Reagent optimization (including primer example. in a given reaction is limited only by the number of • Use primer design software to determine whether available spectrally distinct dyes and the number of dyes any of the primer or probe sequences are prone that can be excited and detected by the real-time PCR to dimerize. Multiplexing requires the concentrations of all components. While more time- program. can analyze all the not interact with each other. Multiplexing a Reagent optimization normalizer and gene of interest in the same tube A critical concern in multiplex reactions is the competition eliminates well-to-well variability that can arise if for reagents among the different targets being amplified. Most fewer reactions to analyze the same number of optimization experiments begin by testing different primer targets. conjugated • Design primers with Tm values within 1°C of each to the fluorogenic probe or primer pair specific for that another. but adjusting primer the amplification of one or more targets. magnesium. Each reaction efficiency. Data analysis 4. Vallone. Mismatched Tm values a normalizer gene and a gene of interest in the same will result in an efficiency bias. multiplexing offers several distinct Thermo Scientific™ website. advantages: • Less variability.5 Multiplex real-time PCR analysis Multiplexing is a technique in which more than one target a segment ranging from 60 bp to 150 bp will enhance is analyzed in the same real-time PCR reaction. it may exhaust the shared components. Designing primers to amplify 46 . Another and thermostable DNA polymerase. Primer and probe design is arguably the most critical factor in a multiplex assay. and fluorophore/quencher reaction components in the early cycles. National Institute of the different primer pairs and/or probes in a reaction must Standards and Technology. Typically. Limiting into consideration. and dNTPs and increasing • Keep amplicons short. As reaction complexity increases. more consistency. In general. is available on the consuming to optimize. target is distinguished by a particular dye. multiplex reactions are used to amplify annealing at one temperature. AutoDimer instrument. performance while minimizing competitive effects: Other alternatives include increasing the amounts of Taq DNA polymerase.

When building a multiplex panel. again trying to achieve close to 100% efficiency. Fluorescent quenchers such as TAMRA™ dye.com 47 . Optimizing the efficiency of the overall multiplex assay. In a multiplex reaction. Evaluate each primer and probe set on an individual basis to determine the designs and conditions that are ideal for the target. it is also advisable to add targets one at a time rather than combining all targets in the first experiment . the choice of quencher for each dual-labeled fluorescent probe becomes more important as the number of probes being multiplexed increases. This is the initial assessment of primer and probe design. There are two main stages in this validation process: 1. This is accomplished through a dilution series standard curve. Using the same standard curve methodology. optics are able to filter the wavelengths so that little. With compatible 90-110%. a standard curve should be completed to assess the reaction efficiencies of 4 all the targets in a multiplex reaction prior to running the assay. work by releasing the energy of the fluorophore at a different wavelength. Data analysis the buffer strength to try to boost the sensitivity and each dilution. Similarly. Choose appropriate multiplex dye combinations based on the detection capabilities of the instrument you are using. The Ct values in the multiplexed reaction should be fluorescence is attributed to the wrong fluorophore (such comparable to those obtained in the singleplex reactions interference is also called cross-talk). there will be little change Fluorophore/quencher combinations between single and multiplex reaction standard curves. Ideally. on the other hand. After each primer/probe combination has been functionally validated. if any.if possible. target with their corresponding singleplex real-time PCR reaction efficiencies. It is important to keep in mind that the singleplex conditions for primer/probe concentrations may not be optimal when the targets are multiplexed. Validating the multiplex assay As with singleplex real-time PCR assays. optimization of the primer/probe concentrations dyes. release energy in the form of heat rather than fluorescence. which can complicate filtering and possibly data integrity. If The reporter fluorophores in a multiplex reaction must be the reaction efficiencies for the multiplexed targets vary spectrally distinct so that the fluorescence signal arising by greater than 5% or fall outside the desirable range of from each is detected in a single channel. 2. combine all primers and probes and perform the multiplex reaction for lifetechnologies. so that the sensitivity is not compromised. Compare the resulting efficiencies for each amplification efficiency of all targets involved. a common quencher for FAM™ dye. Validating the primers and/or probes for each target and determining their individual efficiencies. move on to multiplex optimization. the real-time PCR instrument excitation and emission or other components in the multiplex assay will be required. and therefore keep the overall fluorescent background lower. quenchers of this type result in multiple signals at different wavelengths. Dark quenchers such as NFQ or QSY® quencher.

Troubleshooting 5 .

1 Introduction 50 5.2 Frequently asked questions 59 5 lifetechnologies.com 49 . Troubleshooting 5.

Agarose gel analysis to investigate primer-dimer forma- tion. Formation of primer-dimers Primer-dimers form when partial sequence homology exists Determining if primer-dimers are between the members of the primer pair. If the primers present anneal to each other during the PCR reaction. Before discussing how this is done. itself and therefore set up a competitive environment with primer-dimers often increase. Depending PCR. be performed any time a new gene is being studied or are highly dependent upon primer-dimers being absent. • Storing primers and probes • Real-time PCR inhibition and poor reaction efficiency Although bioinformatics-based primer design can reduce • Software analysis settings the likelihood of dimer formation. assay parameters are altered. 5 the template. it is also possible for a primer to fold upon template annealing and extension. which in turn decreases all lead to confidence in the data and ultimately results sensitivity. it is first important to understand why dimer formation should be minimized or reduced.M. common real-time PCR difficulties AutoDimer (authored by P. there is competition between dimer formation and on its length. The resulting assay will have the highest sensitivity and competition within a reaction well also has a direct impact dynamic range. the Taq DNA Gel electrophoresis is a great way to visualize primer- polymerase may be able to extend them and create a product dimers. This in turn shifts the Ct and standard curve assessment. which analyzes the sequences of primer pairs and flags those that theoretically • Formation of primer-dimers have a tendency to dimerize. it is time well spent. especially that present The downside to gel analysis as the sole method of in multiplex reactions. This could involve adjusting because the dye would bind to them nonspecifically primer concentrations. Problems caused by primer-dimers The effect that primer-dimers can have on a reaction depends largely on the chemistry being employed. The advantage of Primer-dimer formation is one of the most common gel analysis is that the size of the product can help in the problems you will have to troubleshoot during real- time PCR design and validation. In this case. Vallone. assuming proper assay design was free tools available to assist with this. on the other hand. While the extraneous signal is the most important factor. National Institute can be grouped into four main areas: of Standards and Technology. As mentioned earlier. usually below 100 bp (Figure 29). Troubleshooting 5. Dimers appear as diffuse bands at the bottom of the gel. it is still necessary to monitor dimerization experimentally. accepted by the research community. skews results. the nucleic acid sample was Fluorogenic probe-based reactions tend not to be serially diluted and added to the components of a PCR mix. As template decreases.1 Introduction Part of implementing an ideal real-time PCR assay involves annealing and being cleaved in a primer-dimer region is an optimization to ensure that all parameters of the reaction extremely rare event. a high efficiency (which correlates with on reaction efficiency. Primer-dimers appear as diffuse bands near the larger than the original primers and one that is more prone bottom of the gel. There are several daunting. Reactions that rely on accompanied by any required reaction adjustments should double-stranded DNA-binding dyes. However. These factors efficiency shrinks dynamic range. and then confirming the parameters through a monitored during the reaction. thermocycling temperatures and and therefore contribute to fluorescence signal being times. Prior to the thermal cycling reaction. increases the opportunity for these validation is that its sensitivity is in the low nanogram unwanted interactions to occur. Reaction complexity. While optimization does take time. USA). and the influenced as much by primer-dimers because a probe same volume from each mixture was loaded on an agarose gel. During to anneal erroneously as cycling progresses. poor reaction high accuracy). Assay validation is the main factor for consideration. It is best to take simple precautions during primer design Troubleshooting a real-time PCR reaction can seem to avoid dimerization in the first place. competition for primers are finely tuned for accurate results. but many opportunities exist to eliminate them from real-time PCR reactions. One such tool is taken into consideration. and excellent reproducibility. range and therefore may be inconclusive. Figure 29. 50 .

com 51 . but this can be reduced double-stranded DNA-binding dyes. A very specific to 60 nM if necessary. The primer- dimer can be identified in (B) by the signal produced by the NTC sample in the amplification plot. Peak shape and melting temperature 5 toward dimerization. Also 1. tight peak on the 3. In most cases. amplification will result in a single. The first is optimization of the thermocycling note that there are times when primer dimers are not a conditions. Primer-dimers are favored at concentrations plate. hot-start dimers. As mentioned in the data analysis section. primers are designed or if the ΔCt between your samples and the NTC well is >10 so that they anneal successfully at 60°C. a final concentration a standard part of reaction thermal profiles that employ of 200 nM per primer is ideal. as usual.) directly to a 60°C annealing and extension step) lifetechnologies. For example. broader “waves” that indicate melting in 4. are primer may help. more than one primer set for the same target well. compare the observation with the NTC 5. and additional peaks in the melting profile.   overall interpretation when dissociation curve (melt curve) facilitates robust amplification. dissociation curve. In most cases. Reducing or removing primer-dimers If primer-dimers are a concern. temperature. which mainly involves raising the annealing concern. Amplification plots and melting profiles highlighting specific amplification (A) and primer-dimer effects (B). Magnesium is usually best at a concentration of about dissociation curve for each well on the real-time PCR 3 mM. A primer-dimer peak is much more common when should be tested concurrently. And. Primer concentration can always be lowered. data are also available. If primers were not evaluated for their propensity the 70°C range. This can actually template is absent (Figure 30). 2. Primer-dimers manifest themselves as lower- above this. one-step qRT-PCR reactions due to the lower temperature of the RT reaction in the presence of the primer pair. Ideally. evaluate them and consider are attributed to the small and variable sizes of primer- redesigning if necessary. because (since this means that the contribution of any fluorescence two-step cycling (a 95°C denaturation step that goes from the primer dimers to the overall signal is negligible. if there DNA polymerases and reaction setup on ice are also is any doubt whether primer-dimers are present in a preferable. also referred to as melt curves. save much time that would otherwise be spent on optimization if one of the pairs works immediately. and even employing different ratios of forward primer to reverse Dissociation curves. there are many options for Keep in mind that dimers may be more of a concern in reducing or eliminating their occurrence in a reaction. Troubleshooting A B   Figure 30. if they only appear in the NTC wells. fluorescence.

primer and probe storage can have a major effect on the long-term success and consistency of a real-time PCR assay. In many cases. However. which increases background and decreases the signal-to-noise ratio. whether they have undergone prolonged exposure to light. under suboptimal conditions. 5 observing a higher-than-normal level of background fluorescence on the instrument’s multicomponent view is indicative of probe degradation. but not detected by the real-time PCR instrument. Figure 31B are what one would expect from properly stored primers. Figure 31 shows standard curves highlighting the effects of poorly stored primers. If the fluorescent probe or primer is not degraded but the dye itself is. and the composition of the storage solution. storage- related effects may be observed within 6 months. In the case of fluorescently labeled probes and primers. The best method to evaluate primer integrity is consistent employment of standard curves. an ethidium bromide–stained gel can show when product is made. length of time in storage. The main factors that affect primer and probe stability are the storage temperature. Amplification plots showing effects of poorly stored prim- ers (shown by the melt curve and background fluorescence) negatively affected by degraded primers. In assays that rely on fluorescently labeled primers and probes. while the curves in (A) vs. degraded probe releases free dye. making them less detectable in the real-time PCR instrument. Problems caused by poor storage of primers and probes Improper storage of primers and probes can cause them to degrade and lose specificity. are common signs that stability is low. 52 . primers and probes are stable for up to a year (or more). B Determining if primer or probe integrity is compromised The first preventive measure to ensure primer and probe stability is simple monitoring of the storage time. especially if not seen previously. The melt curve provides additional detail. Fluorescent dyes attached to primers and probes can also undergo photobleaching over time. properly stored primers (B). the concentration of the stored primer or probe. Replicate inaccuracy and multiple peaks in the dissociation curve. Troubleshooting Storing primers and probes A Although often overlooked. which in turn affects the reaction efficiency. The amplification plot in Figure 31A is Figure 31. showing that multiple nonspecific products are present. This can manifest as very rough amplification curves due to the low fluorescence.

For example. This can be a random event in which primers.com 53 . in fact. For example. target template) is used to obtain an efficiency value. primer concentration can have an effect on This efficiency value acts as a marker of overall reaction stability. Moreover. magnesium. Use clean work spaces. Here are some steps you can take thermocycling conditions. protect the labels from light (such as the use of opaque a standard curve (generated from a dilution series of the tubes and dark storage) extend their life. Once reaction efficiency reconstituted. efficiencies between a target and a normalizer is quite 2. buffer creates a more stable environment than water. Low efficiency leads to one diagnosis. and high is not recommended. and thus this is often for multiplex experiments). amplification signal the DNA or RNA might inhibit the Taq DNA polymerase. But there is would have the same effect. To avoid contamination from previous PCR reactions. including wiping down inefficient reaction. more desirable than an efficiency of 99% for target A and 92% for normalizer B. If you see amplification in every NTC poor reaction efficiency include primer Tm values being well. especially not every NTC will show amplification. Storing primers at a concentration below 10 μM “health”. lifetechnologies. TE that contains 0. over time especially when plasmid controls are in use (which There are four keys to maintaining primer and probe can be very easily spread but hard to remove). and carryover from nucleic acid purification. 5 surfaces and reagents with nucleic acid–degrading Whether an efficiency for a target is high or low. especially when labeled. Lastly.1 mM EDTA (compared to Causes of high or low efficiency 1 mM EDTA in standard TE) is a good choice because of An efficiency above 110% indicates that inhibition is the sensitivity of some PCR reactions to EDTA that may occurring in the reaction. the importance of reaction efficiency be monitored for signs of decreased functionality beyond a should be well understood among the critical factors in year or so. To determine which reagent is problematic. important for maintaining data accuracy. Causes of inhibition include poor be carried over. to prevent/remove contamination: competition for resources in the tube can produce an 1. chaotropic salts used to bind As discussed in the previous section. set up reactions in a different location. another case in which you may see amplification in NTC Inhibition is normally less common than poor reaction wells. and Taq DNA polymerase. To review. matching solutions as needed. which is an efficiency below 90%. Lastly. Other factors contributing to due to pipeting errors. Causes binding dye–based reactions. Troubleshooting Maintaining primer and probe stability 4. When possible. so that efficiency of 95% for target A and 96% for normalizer B is PCR products from previous reactions are degraded. RNA or DNA quality. if NTC amplification silica columns are employed. primer concentrations of efficiency. then it is likely that one or more of your reagents has more than 5°C different from each other and suboptimal become contaminated. Steps to improve 100  μM are easier to work with in most cases. an use master mixes containing dUTP and UDG. swap out the reagent with a new tube or different source when possible. While the ideal and probes should also be stored in aliquots to minimize reaction efficiency is 100%. For labeled primers and probes. If can be observed in NTC reactions that use double-stranded organic extractions are used. measures that real-time PCR assay design and optimization. As mentioned earlier. high template concentration. For both probe-based and double-stranded DNA efficiency. the widely accepted range freeze-thaw cycles. to a different diagnosis. phenol and ethanol carryover DNA binding dyes when primer-dimers form. primers should be kept at –20°C and should At this point. stability. 3. Lyophilized primers have more flexibility Real-time PCR inhibition and poor with respect to storage time and temperature. Primers these scenarios will be quite different. you can see late amplification include suboptimal reagent concentrations (mainly due to contamination. TE is 90–110%.

Figure 33 shows in mind that any concentrations removed from the the same plot with different baseline settings. 4. 3. even probe ratios. Determining if efficiency is skewed In some cases. Sometimes the process can be relatively pain-free. the assay is fine. The ideal primer concentration can be anywhere from 100 to The problem with skewed efficiency 600 nM. mainly multiplexing reactions. designed to have similar Tm values. In some circumstances. mal curve shapes. Poor efficiency is resolved through assay optimization. As the template becomes more dilute. • Amplification curve baseline linearity • Baseline range settings Resolving poor efficiency or inhibition • Threshold Once it has been determined that the reaction is inhibited • Reference dyes or is operating with poor efficiency. Another solution involves re-purifying the template. assay. there are cases in which the instrument analysis may be masking an otherwise successful assay. will be encountered with the unknown samples and look at the efficiency over that range. which is too wide because fluorescence is detected as early 2. Raising the magnesium concentration as high as 6 mM can improve efficiency in situations where a single product is amplified. Figure 34 shows the baseline reset to the linear 54 . A dissociation curve or gel showing multiple peaks or Analysis settings that play the largest role in data accuracy products means there is a competition for reaction are: resources that almost certainly will have an effect on the 5 reaction efficiency. the best method for determining may in fact be software-related. The result is a curve that dips down and pushes Remember to allow extra drying time to remove the Ct later. Figure 32. Validating and/or whether a particular assay is inefficient is to generate a optimizing software settings can often bring results back standard curve of template diluted over the range of what in line with expectations. However. Troubleshooting ethanol from ethanol precipitations or to employ additional on-column washes to remove chaotropic salts from silica-based purifications. For inhibition. good at automatically setting the baseline within the flat 1. as assay complexity increases. skew results and lead to false conclusions. in cases where a very early concentration of template can be removed and the Ct is observed. mainly because 6. It should be as close to 100% Software analysis settings as possible. Template dilution series to assess reaction efficiency. leading to a smaller dynamic range and decreased versatility (Figure 32). Figure 33A standard curve may not be used during the actual shows a plot with a baseline that spans cycles 1 through 14. optimization can be laborious. those wells with the highest portion of the curve. as cycle 10. a primer and probe optimization matrix is necessary. the baseline can mistakenly be placed back to under 110%. inhibition In this application. but lowering the magnesium may help in cases where competition is   occurring. inhibition and poor efficiency can affect assay the Tm values of the primers. different ratios or concentrations vanishes and the curves take on the more characteristic exponential of forward primer to reverse primer. issues that appear to be reaction-related As mentioned earlier. while probe concentrations can be between Efficiencies outside the range of 90–110% may artificially 100 nM and 400 nM. such as in cases where 18S rRNA is used standard curve reanalyzed. but in other situations. and that the primers are sensitivity. and sometimes phase shape. The instrument software is usually desirable range. Just keep to include a region that is no longer flat. If the efficiency improves as a normalizer. In the annealing temperature) are favorable based on addition. 5. As mentioned. Ensure that the thermal cycling conditions (especially targets for comparison will have different efficiencies. there are some steps The amplification curve baseline linearity is one parameter that can be taken to bring the efficiency value back into the that can affect results. are tested to find the ideal concentration combination for a given assay. Dilutions with earlier Ct values exhibit compressed Ct values and abnor.

A B C 5 D E F Figure 34. the following situation can often occur. range of cycles 2 through 8 and returns the curve and Ct to When evaluating more than one kit or chemistry on the their accurate locations.81. each data set should be studied adjustment can sometimes improve results even further. which is well outside the preferred range of –3.com 55 .   independently so that the ideal threshold may be selected The threshold (the level of fluorescence deemed above for each situation. background and used to determine Ct) is another parameter set automatically by the software. the instrument default settings are more often red plots in that data set because the ideal threshold is than not acceptable for a given assay. The software will automatically select a threshold that is Baseline range settings are not often considered but ideal for the curves with the higher plateau (the blue can have an effect on the reaction efficiency. manual much lower. lifetechnologies. (C and F) Here we see the same curves as in panels A and D but with a baseline that the instrument automatically chose. Amplification plots showing an incorrectly set baseline (A) and a correctly set baseline (B). but one that may also be manually adjusted (Figure 36). and the slope improved even further to nearly 100% efficiency. Troubleshooting A B   Figure 33. However.58 to –3. This would bias the Ct values for the Figure 35 . The slope is now inside the ideal window and the assay is now validated across this dynamic range. As shown in plots in Figure 35).10 (corresponding to 90–110% efficiency). (B and E) These plots were manually adjusted to have the baseline incorporate 4 to 5 additional cycles. Comparison of baseline setting methods to achieve acceptable reaction efficiencies. same run. (A and D) The baseline in these plots is manually set very wide and the slope of the standard curve is poor—only –2. Therefore.

1.9. The most accurate portion of an amplification curve for Ct determination is directly in the middle of the exponential phase when Employing reference dyes such as ROX™ dye and viewing the log plot. Look at the amplification curves in the log plot view and verify that the threshold is set near the middle of the exponential phase of the curve. it is OK to push the limits of detection from very high template to very low template. Standard curve dynamic range validation determines what template concentrations are acceptable in a given assay. knowing that Figure 35. and increase or decrease the baseline range as necessary. if the level of ROX™ dye. Concentrations from the high and/or low ends of a standard curve can also be removed to improve the efficiency of a reaction. if a mix was used that did not have Figure 36. Visually. it is a reference dye normalization issue. it can be seen that the data are actually just fine (Figure 37B). which is outside the desired efficiency window. it is important to make sure that the reference dye 5 outlier data. tial phases. It has become so common that this “behind-the-scenes” factor is often forgotten or assumed not to have a negative impact on a reaction. Improvement of standard curve slope achieved by excluding ROX™. Troubleshooting Again. fluorescein is a powerful method of insulating against some instrument. the threshold can be manually dragged to the middle of the exponential phase for greater accuracy if needed. it seems as if the reaction failed and much optimization is necessary. but where to start? If the ROX™ dye channel is switched off as the normalizer and data are reanalyzed. In general. Across this range. and keep in mind that outliers and whole dilution sets may be removed from the standard curve to improve efficiency and R2 values (as long as those dilutions will not be used when evaluating the “unknown” samples). while the default settings are often very appropriate. Amplification was performed on a dilution series over 5 option in the software is set to None for the analysis. verification of these curve. the default instrument software settings lower panels have had the highest dilutions omitted from the standard are fine in most situations. for example. the software reports the fluorscence signal at Rn (normalized reporter value). as long as those concentrations are never employed during the actual assay (Figure 36). Therefore. which manifest as jagged and inconsistent amplification plots (Figure 37). The curves represented by the As mentioned. The slope has improved to –3. orders of magnitude. However. Adjust the y-axis to be appropriately scaled for the fluorescence plateau intensity. For instruments that employ a reference dye. settings can increase confidence in data accuracy. The ideal threshold setting is sometimes unique for each set of data. However. it is important to understand the relationship between the instrument software and the dye itself. which is considered efficient enough to validate the assay.and user-related errors. Log plot screen shot showing an example of two sets of   terminal data points can always be removed if efficiency is curves with differing plateau heights and therefore different exponen- compromised.   Along the same lines. it can result in very poor target signal returns. 56 . which is the reporter dye signal divided by the reference dye signal. the slope is only –2. and the efficiency reanalyzed. Ensure that the baseline chosen by the software is only in the flat range of the amplification curve. is too high.

It is possible that the assay is reverse transcription. levels in different tissues. problems with ants of the gene of interest.com 57 . Check sequence databases such as NCBI for vari- no amplification include: low expression. check with a cDNA sample. by categorizing Related to low expression. you may need to increase the simple process: sensitivity of your assay. Lastly. or ideally. If you are an assay sitting within an intron sequence will not amplify not sure about the expected level of expression.   Troubleshooting is an inevitable aspect of real-time PCR Problems with reverse transcription assay validation and employment. but if your gene of interest is of low abundance in the for example. other sources of target. Also check where the primers/ Problems with low expression probe are targeting along the sequence. You can also check the type of probe stability reverse transcriptase and primers being used. Check that you are not overloading • Ensure that primer-dimers are not contributing to your qPCR reaction with too much cDNA (max load is 20% signal or poor reaction efficiency v/v). • Verify and adjust instrument analysis settings as Check your reaction components to see if any of these necessary elements can be optimized to improve amplification. it is amplification using a given assay. such as Super- • Understand that efficiencies below 90% will be Script® III Reverse Transcriptase. or the NCBI Unigene database for the EST identical for a particular assay when comparing across a expression data that can give you an estimate of expected data set. which may not be expressed in the samples being studied. designed to only one variant. Is it in a coding The common cDNA input for gene expression is 1 to 100 region or intron? Assays targeting the 5’ UTR of a gene. ng. Additionally. Random • Make standard curve validation the final step in the primers typically yield more cDNA than oligo(dT)-based reaction assessment process methods. Troubleshooting A B   Figure 37. run against a positive control sample to been included in the plasmid for transfection). will not detect an exogenous gene target from sample then you may need to use more. (B) Once the signal from ROX™ dye is removed from the analysis. Likewise. Once you verify that all possible that the primer/probe is not designed to the right the steps above have been addressed. this can be a relatively abundance in your sample. (A) A high level of a passive reference dye such as ROX™ can lead to poor target signal returns. which will also increase your yield. Test a range of a transfected cell (since the UTR region would not have input. Problems with assay design No amplification 5 One final problem that may occur is a complete lack of If you are not seeing any amplification with the assay. as this can introduce inhibitors into the reaction and • Take the steps necessary to maintain primer and thus reduce the efficiency. have been engineered to addressed very differently from values above 110% be more thermostable. Correction for reference dye. However. keep in mind that threshold settings need to be the literature. or assay design. lifetechnologies. if the gene of interest is of low and understanding the key issues. the target signals fall within the expected range. some enzymes. confirm that the assay is functioning properly.

Troubleshooting 5. Ruijter JM. Q: When would I use one-step as opposed to two-step qRT-PCR? Q: I have found that my more concentrated template samples give me less efficient amplification curves: A: Two-step qRT-PCR is popular and useful for detecting Dilute sample gives a slope of –3.000 pg of DNA)/3. and why are they used in real- egg cells have 3. = 3. 58 . Muszta A et al. or non-fluorescent quenchers such as MGB-NFQ. The = 1. thus allowing for the design of shorter. There is one copy of every time PCR? non-repeated sequence per 3. because the ΔΔCt calculation 1 bp = 618 g/mol method works on the assumption that PCR efficiencies 1 genome copy = (3 x 109 bp) x (618 g/mol/bp) are comparable.99. which enables more Acids Res 31:e105. 100 ng of genomic DNA would have: probes so that they can quench the emission from (100. Anal Biochem 302:52–59. In general. Why? However. which emits the light at a different is employed). or Black Hole Quencher® dyes. in the PCR reagents or are not arising from a poorly optimized assay.02 x standard curve method of comparative quantification 1023 [Avogadro’s number]) with efficiency correction can be employed.08 pg). The 5 minimize carryover contamination. concentrated sample gives a slope of –2.08 x 10-12 g Each somatic cell has 6. • Liu W and Saint DA (2002) A new quantitative method A: MGB-NFQ stands for Minor Groove Binder-Non- of real time reverse transcription polymerase chain Fluorescent Quencher. Neurosci Lett quencher? 339:62–66. The MGB moiety increases the reaction assay based on simulation of polymerase Tm of the probe.16 pg of DNA (sperm and Q: What are quenchers. Quenchers are generally used in probe-based assays to extinguish or change the wavelength of the Q: Why do I have to be concerned about the efficiency of fluorescence emitted by the fluorophore when both my real-time PCR assay? are attached to the same oligo.85 x 1012 g/mol) x (1 mole/6. Deprez RH et al. you need to know something about the (higher) wavelength. Since the entire cDNA sample is component) has been diluted below its inhibitory effect. one-step qRT-PCR can provide greater Here are some references that explain this: sensitivity.85 x 1012 g/mol = (1. since tubes do reason you get better efficiency with the more diluted not need to be opened between cDNA synthesis samples is because the inhibitor (salt or some other and amplification.84. It also 0. chain reaction kinetics.000 copies. are observing are not being influenced by contaminants QSY®. more specific probes. accurate allelic discrimination and makes for a more sensitive real-time PCR assay. This is why you should optimize your system before trying to quantify unknown samples.08  pg of human DNA. When A: If you want to compare the expression levels of two the fluorophore gets excited it passes on the energy genes (for example. amplified. one-step qRT-PCR is easier to use when processing large numbers of samples and helps A: Something in your sample is inhibiting the PCR.2 Frequently asked questions Q: How many copies are in a given amount of human Q: Can I compare Ct values of PCR reactions with different genomic DNA? efficiencies? A: 1 genome copy = 3 x 109 bp A: You should not compare Ct values of PCR reactions with different efficiencies. A: Quenchers are moieties attached to primers or Therefore. They usually do this by fluorescence resonance energy transfer (FRET). down to 0. Nucleic matched and mismatched probes. • Ramakers C. (2003) Kinetic probes exhibit great differences in Tm values between outlier detection (KOD) in real-time PCR.08 pg = ~33. the TaqMan® MGB • Bar T. (2003) Assumption-free analysis of quantitative real-time Q: What is MGB-NFQ? What is the benefit of it as a polymerase chain reaction (PCR) data.4 and an R2 value of multiple messages from a single RNA sample. in cases where a normalizer gene to the quencher. 1 ng of a fluorophore that is also attached to that primer or DNA has 330 copies.5 and an allows the archiving of cDNA for further analysis. Common quenchers are TAMRA™ efficiencies of the PCR to confirm that the Ct values you dye. R2 value 0.1 pg total RNA. probe. Ståhlberg A.

com 59 . Troubleshooting 5 lifetechnologies.

Advanced topics: digital PCR 6 .

Advanced topics: digital PCR 6.3 Digital PCR applications 65 6.1 Digital PCR 62 6.com 61 .2 Digital PCR attributes 64 6.3 Beginning a digital PCR experiment 68 6 lifetechnologies.

000 partitions. if you have a viral DNA sample and a digital compatibility. curve. all without reference to standards or controls (Figure 38). is that with the fraction of negative reactions is used to generate an more reactions. by chance. the ratio of positive and PCR. and you split the mixture into 20. In this example.000 digital PCR reactions The QuantStudio® 3D Digital PCR System leverages a chip. or a few molecules. Advanced topics 6. while other partitions do not. there would be differences are detailed in Table 1. respectively. a significant number of reactions that contain zero. a sample is viral target. QuantStudio® 3D Digital PCR 20K Chip. The primary difference between real-time PCR reagent mixture that contains 20.000 individual PCR reactions. if 20% of the 20. These key attributes are driven by the number of Figure 38 shows the Poisson distribution model. partitioned into thousands of individual PCR reactions— mathematically you would expect to have approximately 1 in essence generating a limiting dilution. the answer will be the same regardless of the Following amplification on a dual flat-block thermal cycler. optimally partitioning a standard PCR can be identified by finding 20% on the x-axis and identifying reaction mix into 20. number of reactions. The difference. 6 Figure 38. or time PCR. Since the calculation is based on a leading to positive and negative reactions. however. percentage. 62 . TaqMan® Assays are ideally suited to perform negative signals will follow a classical Poisson distribution. Continuing partitions and volume sampled in the digital PCR reaction. fluorescent statistical analysis. the confidence interval is narrower so the absolute count of the number of target molecules in the statistical reliability of the data is improved. SYBR® Green dye has demonstrated For example. digital PCR. sample. Other key copy in each reaction.59 copies/reaction. is 1. Each chip is designed with 48 subarrays x 64 through-holes/subarray. one. gave a negative signal. digital PCR offers a highly precise and sensitive more than two copies—the probability of these outcomes is approach without the need for a reference or standard described by the Poisson model.1 Digital PCR Digital PCR compared to traditional Target quantification in digital PCR real-time PCR Quantification by digital PCR is achieved using fairly simple Digital PCR employs the same primer sets. Upfront the corresponding average copies per reaction based on sample dilution ensures that a portion of these partitions the dashed line on the graph. and enzymatic reagents as traditional real-time contain zero. Figure 39 shows the basic procedure used in digital PCR. however.000 copies of your PCR and digital PCR is that in digital PCR. the result contain the target molecule. two. Hydrophilic and hydrophobic coatings on plates enable reagents to stay in the bottomless through-holes via capillary action. Since each reaction is expected to labels. In contrast to real. with our example. Of course. the number of target copies in each based technology.

ΔCt. Digital PCR employs a simple workflow and uses familiar techniques.g. lifetechnologies. TaqMan Assays or ® Results are not affected by any of these SYBR® Green dyes) factors Real-time PCR instrument used Amplification efficiency of PCR primers/probe Table 1.com 63 . Advanced topics: digital PCR Prepare sample Partition sample Amplify DNA Derive answer Copies/µL Sample partitioned Positive reactions into many reactions Negative reactions Figure 39.. Conventional qPCR Digital PCR Output from experiment Ct. Poisson equation used to calculate target quantity from digital PCR data. 6 Figure 40. Comparison of conventional qPCR to digital PCR. or ΔΔCt Copies per µL Quantification Relative quantification Absolute quantification Results can be affected by the: Detection chemistry (e.

In the example in Figure 42B. Increasing replicates increases the statistical performance of current TaqMan® real-time PCR assays in order to drive additional specificity. significance of your answer. and precision? Figure 41. specificity. Advanced topics 6. number of replicates for the target. and increased specificity with the ability to perform absolute quantification without a standard curve. the reaction wells reach a point where the wild type signal no longer overwhelms the mutant signal. a sample containing 99 wild type molecules and 1 mutant equates to the mutation being present at 1 in 100 or 1%. effectively decreasing background noise. For maximum precision. By individualizing the reaction. The chances that you capture a particular ball of interest from the container is increased with the greater amount of balls taken out of the container. 1:5 Partitions Precision is driven by the number of replicates that Figure 42. For example. How does digital PCR manage this extra sensitivity. By first partitioning the sample. digital PCR enables us to extend the performance of current TaqMan® real. competing wild type sequences in any reaction containing a mutant are reduced. By individualizing the reaction. Increasing the amount sampled from the actually not that different from real-time PCR. A time PCR assays in order to drive additional specificity. digital PCR extends the are run. the single mutant is lost in a sea of wild type copies (Figure 42A). Imagine a container full of balls (Figure 41). however. The Poisson principle assumes an appropriate volume of Sensitivity is driven by total volume interrogated. the percentage of negative reactions should be targeted between 5% and 80%. thereby giving more 6 confidence that the value determined represents the actual target quantity in the sample. Specificity is driven by the assay and number of replicates run. It’s the total pool is sampled. dividing our sample into twenty digital partitions reduces the sample complexity within each partition to 1 in 5 or 20%—theoretically a twenty-fold improvement compared to the starting sample. 64 .2 Digital PCR attributes Detection of low levels of pathogen Digital PCR extends the performance of TaqMan® Assays by enabling additional attributes that go beyond the limits of real-time PCR. If sufficient partitions are B used. total pool increases the ability to accurately determine the number of digital PCR yields a statistical perspective via its large targets. Using TaqMan® SNP Genotyping Assays in standard real-time PCR mode. increased sensitivity. These attributes represent three main categories­—increased precision. giving greater visibility to whether or not you are able to detect the target of interest.

Studio® 3D system and a standard TaqMan® Copy Number and thus are an important area for detailed study.2-fold difference (Figure 43B).95 0. was analyzed using the Quant- CNVs are linked to susceptibility or resistance to disease. real-time PCR (qPCR).98 0. confirming that digital PCR cases.06 N/A NA17251 6 1 0. measurements are not sufficiently precise for can differentiate less than a 1. The CV (column 6) was below 2. A statis- tion (aCGH).47 NA17132 6 3 2.00 0. Many Assay specific to the CCL3L1 genetic locus found on the long methods of CNV detection exist today. (A) Copy number was measured across 9 DNA samples.com 65 . Replicate measurements cent in situ hybridization (FISH). Advanced topics: digital PCR 6.11 0.07 1.55 B 9 8 7 Copy number 6 5 4 6 3 2 1 0 0 5 1 8 2 4 7 0 2 4 4 5 5 3 9 0 1 0 5 72 72 72 71 91 85 71 72 88 A1 A1 A1 A1 A1 A1 A1 A1 A1 N N N N N N N N N Samples Figure 43.3 Digital PCR applications Precise copy number variation between the target and reference are very small. inversions. or 8.02 2. arm of chromosome 17.98 0. array comparative genomic hybridiza. determining copy number differences where the ratios A Number of Expected copy Detected copy Standard Sample replicates number number (mean) deviation CV (%) NA17245 6 0 0. and 8 copies was clearly discernable as a result of the high Despite advances in some of these technologies.85 NA19194 8 4 4.08 0. tions from 0 to 8 copies per genome (Figure 43A).02 NA18854 8 8 7.13 2. lifetechnologies.12 2.22 NA18507 8 5 5.07 NA17202 8 7 7. procured translocations can lead to biallelic or multiallelic CNVs.20 2.05 2.96 0.21 NA17258 6 2 1. Digital Copy number variation (CNV) is defined as a modification in PCR. (B) As demonstrated by non-overlapping error bars. or A representative panel of 9 genomic DNA samples.6% for each set of replicates. in many degree of precision achieved. the achieved measurement precision enables statistical discernment of the CCL3L1 copy number in samples containing 7 and 8 copies. enables low-percent copy number differences to be sequence differs from a reference or standard.02 0. and next-generation tically significant difference between samples containing 7 sequencing (NGS).06 1. including fluores. from the Coriell repository. Genomic detected and accurately quantified. demonstrating a high degree of measurement reproducibility within each replicate group. deletions. a technology capable of highly precise measure- the genome where the number of copies of a genomic DNA ments.91 0.05 1. comparative genomic indicated that the samples represent copy number varia- hybridization (CGH). alterations such as insertions.50 NA17110 8 6 5. Precision demonstrated for copy number analysis of the CCL3L1 genetic locus on chromosome 17. 6.

Because each data point is generated digitally. Note sequencing of samples. For more information about this the recipient’s allele starts to reappear after 101 days. such as TaqMan® correlation between input concentration and measured concentration. are Figure 44. a TaqMan® Assay. 66 . Two alternate alleles that differentiated a bone marrow donor from a recipient were chosen. This method enables accurate and precise library quantification. a crit. with digital PCR methodology. Ultimately. able to detect mutant cell prevalence down to 1%—and below (Figure 44). is an important aspect of tumorigenesis. sequencing costs by ensuring an accurate quantifica. minimizing the need to re-run or repeat transplant (pre-SCT) and at the indicated times after transplant. such as onco- genes or tumor suppressor genes. they require an assay that delivers high signal-to-noise and low false-positive- to-false-negative rates.9968 such as cancer research because the accumulation of 10 mutations in crucial regulatory genes. the linear slope indicates that the amounts of mutant allele were Assays. To achieve this high degree of precision.1 1 10 Common SNP genotyping technologies. reducing competing wild type sequences in any reaction containing a mutation and effectively decreasing background noise. such as capillary Concentration input (%) electrophoresis (CE) sequencing and real-time PCR. most effective at detecting mutant cells with a prevalence Differing amounts of DNA from three different oncogenic KRAS alleles no lower than about 20% (or approximately 1 in 5 cells). go to lifetechnologies. the reaction wells reach a point where the wild type signal no longer overwhelms the mutant signal. indicating a relapse. Acquisition of these mutations in a tiny 1 subset of somatic cells can be sufficient for cancer initia- tion or progression. can be calculated and a ratio determined (Figure 45).0436 Rare mutation detection has great implications in areas Concentration measured (%) R2 = 0. using 6 digital PCR to quantify NGS libraries decreases overall Figure 45. Advanced topics 100 Rare-allele detection y = 1. This approach limits quantification to library constructs Q8092: +101 days Q8133: +118 days that contain both adapter sequences. allowing for maximizing sequencing yields downstream. researchers are now accurately measured. Allelic chimerism in bone marrow transplant samples.1 Since these mutations are so rare. mutant and wild type. Samples were collected pre-stem cell tion upfront. Note the excellent combining real-time PCR chemistries.01 0. 0. Q7938: +25 days Q7960: +41 days ical step in both the Ion Torrent™ and other NGS workflows.com/dpcrngs 118 days. and is obvious by application. 0. Q7832: Recipient Pre-SCT Q7823: Donor Absolute quantification of next- generation sequencing libraries Next-generation sequencing (NGS) libraries can be quanti- fied with minimal sample handling and without the need to generate a standard curve using digital PCR. If sufficient partitions are used.01 0. Digital PCR works by partitioning a sample into many indi- vidual reactions prior to amplification. is available. By were spiked into a constant amount of normal DNA. designed to span both the forward and reverse adapters specific to each library.1006x + 0. the total count of each allele. Rare allele measurement using spike-recovery method.

6 lifetechnologies. with extremely tight precision for each dilution. digitally measured assay standards can enable laboratories to compare results. For many organisms or applications. and results determined in absolute copies per microliter by digital PCR. Copies per reaction for each dilution were calculated and demonstrate excellent correlation (0. (B) For sample 600-T.0 initially be calibrated. Furthermore. Through direct copy number determi- nation. and whether there is sufficient reference material for completion of 0.001 For some studies.0 Copies/µL (x1010) metrology.5 of reference standards using conventional real-time PCR requires consideration of how the reference sample will 1. 0. such as a housekeeping gene like actin. This capability is especially useful for calibrating reference samples and assay standards 10-6 10-7 10-8 10-9 when none exist. Advanced topics: digital PCR Absolute quantification of nucleic acid A standards Accurate genetic measurements often require comparison 2. there is often no suitable reference sample available. it can be used for absolute quantification.01 limited to detecting changes that vary by two-fold or more.5 all future studies. this approach is generally 0. The tight error bars demonstrate very high precision in the measurement of each sample. however. Generation 1.com 67 .9991 1 Copies/reaction Low-level fold change of gene expression 0. an additional 10-fold dilution series covering four logs of dilution was constructed.5 to reference samples and assay standards. it is often 0.0001 10-6 10-7 10-8 10-9 necessary to express differential gene expression with Dilution from stock respect to a reference gene. Figure 46. its long-term stability. In addition. with the assurance that 10 measurements are based on the same absolute baseline.1 Real-time PCR is commonly used to detect differen- tial gene expression. R2 = 0. 600-A 600-C 600-G 600-T Digital PCR does not rely on a reference sample or assay standard. Standardiza- tion of references is especially important in the field of 2.9991). detection of expression changes less than two-fold may be required. (A) Four standards were measured in duplicate. B measuring the exact copy number of a nucleic acid target of interest (Figure 46). the lack of broadly adopted 0 standards impacts comparison between laboratories. Digital PCR precisely and accurately quantifies standards without the use of a standard curve.

com/dpcrcommunity. which seamlessly integrates into your digital PCR gene expres- In addition. use this value to calculate the number per genome. while real-time qPCR was not able to discriminate even a 10% difference between sample 1 and sample 3. Please refer to the your samples is known. Advanced topics With the ability to achieve highly precise measurements of conversion of RNA to cDNA. sample 5. Tukey-Kramer HSD test was done within JMP software with experiment replicates. when QuantStudio® 3D Digital PCR System Experimental Design loaded on a QuantStudio® 3D Digital PCR 20K Chip. After reverse transcription.0 80% -1.6 copies of the target sequence. quantification of a transcript obviates the need for a refer- ence gene. Digital PCR with the QuantStudio® 3D system is able to discriminate a 5% difference between sample 1 and 2 (indicated by non-overlapping circles by Tukey-Kramer HSD test). the sample must be sample would be diluted down to 600 copies/μL or 1.98 diluted such that each reaction contains one or zero mole.2 ∆Ct RQ 70% –1. we offer the of two-fold or less (Figure 47).4 Beginning a digital PCR experiment To perform a digital PCR experiment. digital PCR is capable of resolving changes sion is important to experimental sensitivity. volume of sample needed to target 20. product manual to learn how to determine target copy tion into copies/µL. If the target copy number per genome of 6 setting up a digital PCR experiment. Samples 1 through 5 are mixtures of synthetic miRNAs hsa-miR-19b and hsa-miR-92 at different ratios: sample 1. a 865 pL reaction well volume.05 Sa Sa Sa Sa Sa Sa Sa Sa Sa Sa Sample Sample Figure 47.6 to This can be found at lifetechnologies. 50%.4 60% –1.6 50% –1. sample 4. 100%. sample 2. the ability of digital PCR to determine absolute sion workflow. Like real-time PCR. digital PCR requires the A B 100% –0. 6. ng/μL in the final reaction to give 0. For example. High-Capacity cDNA Reverse Transcription Kit. 90%. cDNA was measured by qPCR and digital PCR on the QuantStudio® 3D system. dilute the samples so that. ΔCts of qPCR between hsa-miR-19b and hsa-miR-92 were reported for each sample. 75%.05 m m m m m m m m m m 0. each Guide for more detailed application-specific instructions. Since the efficiency of conver- ±10% or better.3 pg/copy of a given gene are present per genome and lifetechnologies.8 40% –2.6 copies per reaction cules. the stock gDNA in a given 68 . sample 3. The relative quantitations for digital PCR results were reported in percentile for each sample. Next. Quantitation precision comparison between digital PCR (A) and real-time qPCR (B). Refer to the QuantStudio® 3D Digital PCR System a spectrophotometer and convert the ng/µL concentra. through-hole reaction will contain approximately 0. First establish the starting concentration using well.com/quantstudio3d 1.0 All Pairs All Pairs 1 2 3 4 5 1 2 3 4 5 Turkey-Kramer e e e e e e e e e e Turkey-Kramer pl pl pl pl pl pl pl pl pl pl 0. 95%.8 90% -1. assuming or on the community in the digital PCR forum at 3.000 copies/chip for Each application has its own set of factors to consider when each sample.

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