REAL-TIME PCR

AUTOMATIONS
DAYLE DANIEL G. SORVETO, RMT
LEARNING OBJECTIVES:
at the end of this topic the learner’s should be able to:

UNDERSTAND THE IMPORTANCE DEFINE DIFFERENT APPLY THE PRACTICAL USE OF

OF RT-PCR IN MOLECULAR TERMINOLOGIES USED IN RT-PCR DIFFERENT SOFTWARE ANALYSIS
DIAGNOSTICS IN RT-PCR IN CDCDC LAB
LEARNING OUTLINE:

 OVERVIEW OF REALTIME PCR

 BASIC TERMINOLOGIES
 APPLICATION DISCUSSION OF MA6000 (SANSURE THERMAL CYCLER)
 APPLICATION DISCUSSION OF DESIGN AND ANALYSIS SOFTWARE (VER 1.5 AND VER 2.4)
OVERVIEW OF REAL-TIME PCR

 The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology.
 Using PCR, specific sequences within a DNA or cDNA template can be copied, or “amplified”, many thousand- to
a million-fold using sequence specific oligonucleotides, heat stable DNA polymerase, and thermal cycling.

 In real-time PCR, the amount of DNA is measured after each cycle via fluorescent dyes that yield increasing
fluorescent signal in direct proportion to the number of PCR product molecules (amplicons) generated.
 DATA IS COLLECTED IN THE EXPONENTIAL PHASE OF REACTION
THE ADVANTAGES OF REAL-TIME PCR INCLUDE:

I. Ability to monitor the progress of the PCR reaction as it occurs in real time
II. Ability to precisely measure the amount of amplicon at each cycle, which allows highly accurate
quantification of the amount of starting material in samples
III. An increased dynamic range of detection
IV. Amplification and detection occurs in a single tube, eliminating post-PCR manipulations
 By plotting fluorescence against the cycle number, the real-
time PCR instrument generates an amplification plot that
represents the accumulation of product over the duration of the
entire PCR reaction

Relative fluorescence vs. cycle number. Amplification plots are created when the fluorescent signal
from each sample is plotted against cycle number; therefore, amplification plots represent the
accumulation of product over the duration of the real-time PCR experiment.
REAL-TIME PCR STEPS

 There are three major steps that make up each cycle in a real-time PCR reaction. Reactions are generally run for 40
cycles.
1. DENATURATION: High temperature incubation is used to “melt” double-stranded DNA into single strands and loosen
secondary structure in single-stranded DNA.
1. The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be
increased if template GC content is high.
2. ANNEALING: During annealing, complementary sequences have an opportunity to hybridize, so an appropriate
temperature is used that is based on the calculated melting temperature (Tm) of the primers (5°C below the Tm of the
primer).
3. EXTENSION: At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to
100 bases per second.
1. When an amplicon in real-time PCR is small, this step is often combined with the annealing step using 60°C as the temperature.
QRT-PCR METHODS
TWO-STEP QRT-PCR
 Two-step quantitative reverse transcriptase PCR
(qRT-PCR) starts with the reverse transcription of
either total RNA or poly(A)+ RNA into cDNA using
a reverse transcriptase (RT)
 This first-strand cDNA synthesis reaction can be
primed using random primers, oligo(dT), or gene-
specific primers (GSPs).
ONE-STEP QRT-PCR
 One-step qRT-PCR combines the first-strand Cdna
synthesis reaction and real-time PCR reaction in the
same tube, simplifying reaction setup and reducing
the possibility of contamination.
 oligo(dT) or random primers will generate
nonspecific products in the one-step procedure and
reduce the amount of product of interest
 TERMINOLOGIES

Baseline
• The baseline of the real-time PCR reaction refers to the signal level during the initial cycles of PCR, usually cycles 3 to 15, in which there is little
change in fluorescent signal. The low-level signal of the baseline can be equated to the background or the “noise” of the reaction.
• The baseline in real-time PCR is determined empirically for each reaction, by user analysis or automated analysis of the amplification plot. The
baseline should be set carefully to allow accurate determination of the threshold cycle (C t), defined below.

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.
• It is set to distinguish relevant amplification signal from the background. Usually, real-time PCR instrument software automatically sets the threshold at 10
times the standard deviation of the fluorescence value of the baseline. However, the positioning of the threshold can be set at any point in the exponential
phase of PCR.
Ct (threshold cycle) or crossing point (Cp)
• The threshold cycle (Ct) is the cycle number at which the fluorescent signal of the reaction crosses the threshold.
• The Ct is used to calculate the initial DNA copy number, because the C t value is inversely related to the starting amount of target.
• For example, in comparing real-time PCR results from samples containing different amounts of target, a sample with twice the starting amount will
yield a Ct one cycle earlier than a sample that contained half as many copies of the target prior to amplification.
• As the template amount decreases, the cycle number at which significant amplification is seen increases.

Reporter signal: Fluorescent signal that is generated during real-time PCR by either SYBR Green or
a fluorescently labeled sequence-specific probe.

Normalized reporter signal (Rn): This is the emission intensity of the reporter dye divided by the emission intensity of the passive
reference dye measured in each cycle.

Internal control: This is a control sequence that is amplified in the same reaction as the target sequence and detected with a different probe (i.e.,
duplex PCR is carried out). An internal control is often used to rule out failure of amplification in cases where the target sequence is not detected.
Positive control: This is a control reaction using a known amount of Endogenous: A region of DNA or RNA that always is found naturally
in a test sample and which differs from the target sequence of a PCR
template. A positive control is usually used to check that the primer set or primer–
method.
probe set works.
Exogenous: A region of DNA or RNA that is added to a test sample as
No template control (NTC): This is a control reaction that contains a control.
all essential components of the amplification reaction except the template. This
enables detection of contamination. Inhibition: The reduction in efficiency in a PCR caused by elements
in the environmental sample that interfere with the normal reaction.
Amplicon: Amplified fragment of DNA obtained through PCR.
Aliquot: A smaller volume withdrawn from a greater volume.

Carry-over contamination: The contamination of a sample, or

series of samples or reagents from nucleic acids from previous
amplification reactions (usually carried by the equipment or analyst).
Master mix: Solution containing all reagents (except for the test or sample Reverse transcriptase: A polymerase that catalyzes the
nucleic acid) that is required to perform PCR.
Matrix spike: A QC sample prepared by adding a known amount of target
analyte to a specified amount of sample matrix for which an independent estimate
of target analyte concentration is available. A matrix
spike is used, for example, as a positive control determining the effect of the
matrix on the overall method's recovery and to verify it does not have an
inhibitory effect on the PCR.
Rnase: An enzyme that catalyzes the hydrolysis of RNA.
Primer: A segment of DNA or RNA that is complementary to a given DNA
sequence and that is needed to initiate replication by DNA polymerase.
Primer dimer: Two primer sequences that form a small region of
double stranded DNA due to complementary sequences being present on
both primers. This formation inhibits the primers use in the polymerase
chain reaction.
formation of complementary DNA (cDNA) from an RNA template.
Thermocycler: Device which can be programed to raise and lower
the temperature of reaction mixes and is used to perform PCR.
RT-PCR: Reverse Transcriptase-Polymerase Chain Reaction. A
technique for amplifying RNA, such as 16S rRNA or mRNA, in vitro by
using reverse transcriptase and RNA as a template to create a cDNA that is
then be used in a PCR.
PCR: Polymerase Chain Reaction. A technique for amplifying DNA
sequences in vitro by separating the DNA into two strands and incubating it
with nucleotides, oligonucleotide primers, and DNA polymerase. It can
amplify a specific sequence of DNA by more than a billion times (e.g., 40
cycles = 240 = one trillion amplicons).
 THE PCR SOFTWARE WILL BE DISCUSS IN ACTUAL
DEMONSTRATION BY USING THE SOFTWARE OF (MA6000
PLUS AND QUANT STUDIO 5 MACHINE
 REFERENCES:
• TaqMan Gene expression Assays Protocol (2010), applied Biosystems,
Life technologies corporation
• Menezes A., Troubleshooting qPCR: what are my amplification curves
telling me (2015), Integrated DNA technologies.
• Aboytes R., Berger, P et al (2004)Quality Assurance/Quality Control
THANK YOU
Guidance for Laboratory Performing PCR analyses on Environmental
Samples. U.S Environmental Protection agency
• QIAGEN (2010) Critical factors for successful Real-time PCR
• Real-time PCR handbook(2018) life technologies