Troubleshooting QPCR:: What Are My Amplification Curves Telling Me?

Troubleshooting qPCR:
What are my amplification curves telling me?

Aurita Menezes, Ph.D., Scientific Applications Specialist
Integrated DNA Technologies
Click icon to add picture

Overview
 Basics of an Amplification Curve

 Phases of an amplification curve
 Terminology
 Setting the correct baseline and threshold
 Problematic qPCR Curves

 No amplification
 Unexpected efficiency
 Delayed and early Cq
 Scattered replicates
 Unusual curves
 Noisy signal
 Amplification beyond plateau
 Negative curves
Aurita Menezes
Basics of an Amplification Curve
Background
Aurita Menezes
R, ΔR, Rn, and ΔRn
R= Multicomponent view
∆R= Fluorescence
(fluorescence obtained- Baseline
without any normalization)
Rn: ΔNormalized reporterfluorescence
Rn = Rn – baseline signal=emission of the reporter dye
emission of the passive reference dye (ROX)
Aurita Menezes
Baseline and Threshold
Linear View Log View
 Baseline stop value should be set 1 to 2 cycles before earliest amplification

 Set Baseline in Linear View
 Set Threshold in Log View
Aurita Menezes
Improper Baseline and Threshold
Linear Rn View Log Baselined ΔRn
Aurita Menezes
Problematic qPCR curves
Aurita Menezes
NoNo Amplification
amplification
 Incorrectly assigned dye detector

 Make sure instrument setting for dye FAM incorrectly assigned as TAMRA
matches dye used in probe

 Missing a master mix component
 Repeat the experiment
 Sample degradation
 Does a different cDNA prep give
you the same result?
 Lack of target in sample FAM incorrectly assigned as TET
 Test a positive control
 Assay design
 Try a different assay
 Machine not calibrated for dye
 Calibrate the instrument
Aurita Menezes
Unexpected PCR Efficiency
 Lower efficiency (<85%)
 Incorrect dilutions causing errors in standard curve
 Not enough dynamic range of standard curve
 Primers designed on a SNP site
 Lower fluorescence of dye
 Instrument not calibrated for dye
 Sample inhibition
 Higher efficiency (>110%)

 Incorrect dilutions causing errors in standard curve
 Not enough dynamic range of standard curve
 Genomic DNA contamination
 Incomplete DNase treatment
Aurita Menezes
PCR Efficiency
· Efficiency reflects whether DNA doubled
every cycle
· It takes 3.32 cycles for DNA to be amplified
10 fold
· If samples have been correctly diluted, every
10-fold dilution should be 3.32 cycles
Aurita Menezes
Unexpected PCR Efficiency…..Incorrect dilutions
114%
Template conc. too high
Incorrect
dilutions
100%
Aurita Menezes
Delayed Cq
 Decreased efficiency
 Low expression
 Sample inhibition
 Incorrect normalizer concentration
 Master mix differences
Aurita Menezes
Delayed Cq……..Lower efficiency
 If 10-fold dilutions are all >3.32 cycles apart:

 Are your primers on a SNP site?
 Consider using IDT PrimeTime® Predesigned Assays designed to avoid SNP
sites through the use of updated sequence information from NCBI databases
Aurita Menezes
Delayed issues
Efficiency Cq……Lower fluorescent dye intensity combined with suboptimal instrument optics
for Dye B
Dye A
Dye B
Aurita Menezes
Delayed Cq……Sample inhibition
 The concentration of inhibitors is maximum in the least dilute

sample
 As the sample is diluted, the inhibitory effect decreases
 Make a new cDNA prep, try to minimize contamination with phenol layer
during RNA isolation
10-fold dilution
Aurita Menezes
Delayed Cq……Master mixes can make a difference
Master Mix A
Master Mix A
Master Mix B Master Mix B
10-fold dilutions
HPRT TBP
Aurita Menezes
Early Cq…..Too much template
 Too much template
 Cq value comes up before cycle 15
 True amplification is observed when analyzed in the linear view
Aurita Menezes
Early Cq…..Automatic baseline failure
When too much template is present, it’s likely that the instrument’s
software is unable to distinguish between noise and true amplification.
In such cases, auto baseline may assign an incorrect value for the
baseline correction factor.
 Adjust the baseline manually to correct this problem
Aurita Menezes
Scattered Replicates
 Pipetting errors
 Poor thermal calibration (thermocycler is raising and lowering
temperature inconsistently across different wells)
 Denaturation time is too short (if using a fast cycling master mix,
consider increasing denaturation time from 5 to 20 sec.)
 Low copy number
 Incorrectly set baseline
Replicates ideally should not be

more than 0.5 Cq apart
Aurita Menezes
Height of Amplification Curve
 Lowered background
 Probe concentration
 Signal bleed over
 Incorrectly assigned detector
 Increased ROX in samples
 Master mix
Aurita Menezes
Height of Amplification Curve…..
 Lowered background due to improved quenching

 IDT double-quenched ZEN™ probes (available with IDT PrimeTime®
qPCR Assays) have lower background and increased sensitivity
Aurita Menezes
Height of Amplification Curve……Incorrect probe concentration
Correct Probe
Concentration
Incorrect Probe
Concentration
Aurita Menezes
Height of Amplification Curve…. Amount of ROX
50 nM ROX
50 nM ROX
100 nM ROX
Noisy signal
10 nM ROX
Aurita Menezes
Height of Amplification Curve……Multiplex vs. Singleplex
 The height of amplification curve is typically lowered when a target is
investigated in a multiplex reaction vs. a singleplex reaction.
 More importantly, it is critical that the Cq is not shifted between both
reactions.
If multiplexing,
 The master mix needs to be
adjusted for additional
dNTPs, Mg, and Taq enzyme
Singleplex
or
Multiplex
 Use a master mix
specifically designed for
multiplexing
Aurita Menezes
Unusual curves……….Sample evaporation
Aurita Menezes
Unusual
Noisy curves…………
signal- too much probe
Too much probe (6X)
Aurita Menezes
Unusual curve…….Negative curves
 Dye calibration issues on instrument
Aurita Menezes
Unusual curve……..Negative curves
 If the instrument is not correctly calibrated,
 Fluorescence due to amplification increases in a given channel, however the fluorescence
attributed to background will also increase, while fluorescence attributed to the other
dyes and the normalizer may be artificially lowered resulting in negative curves
 Calibrate the machine

again for all the dyes
being used
Aurita Menezes
Unusual curves….Amplification beyond plateau
Aurita Menezes
Unusual curves….
 Amplification is observed beyond plateau

 Fluorescence detected is at maximum capacity for the detector
 Consequently, the amount of fluorescence attributed to Rox is
mistakebly decreased as the amount of fluorescence attributed to
When ROX normalization is
back ground increases.
turned off,
 Consequently fluorescence
the curve looks normalis normalized to a smaller Rox value,
artificially increasing the heinght of the amp curve
 Turn normalizer off
Aurita Menezes
Summary
 Information on PrimeTime® qPCR Assays with ZEN™ double-quenched probes and

PrimeTime® qPCR Primers can be found at:
http://www.idtdna.com/pages/products/gene-expression/primetime-qpcr
 For background on setting up qPCR experiments, qPCR protocols, and troubleshooting

information like that presented in this webinar, download the IDT PrimeTime® qPCR
Application Guide at:
http://www.idtdna.com/pages/support/technical-vault/reading-room/user-guides-protocols
 Information on products that can be used as controls such as MiniGenes™, gBlocks™,

Ultramers™ and can be found at:
http://www.idtdna.com/pages/products/genes/custom-gene-synthesis
http://www.idtdna.com/pages/products/genes/gblocks-gene-fragments
http://www.idtdna.com/pages/products/dna-rna/ultramer-oligos
Aurita Menezes
Aurita Menezes
Unexpected Signal…
 Positive NTC -> maybe master mix got contaminated with template
during qPCR prep
 Positive –RT -> gDNA contamination
 Incomplete DNase treatment
 Assay design
Aurita Menezes
Threshold
Linear Scale
Logarithmic Scale
Bad Threshold – Good Threshold – Bad Threshold –
in plateau phase in exponential phase in baseline phase
Aurita Menezes
Ideal Standard Curves
102%
Aurita Menezes
Height of Amplification Curve….. Level of ROX
Least ROX ROX Normalization =OFF
High Rox
ROX Normalization=ON
Aurita Menezes
Unusual Curve…..Complete evaporation of sample
Aurita Menezes
Scattered Replicates...Low copy number
Aurita Menezes
Delayed Cq…..High ROX in reaction
 Differences in ROX concentration
Aurita Menezes

Troubleshooting QPCR:: What Are My Amplification Curves Telling Me?

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Troubleshooting QPCR:: What Are My Amplification Curves Telling Me?

Uploaded by

Copyright:

Available Formats

Troubleshooting qPCR:

What are my amplification curves telling me?

Click icon to add picture

 Basics of an Amplification Curve

 Problematic qPCR Curves

Linear View Log View

 Baseline stop value should be set 1 to 2 cycles before earliest amplification

Linear Rn View Log Baselined ΔRn

 Incorrectly assigned dye detector

matches dye used in probe

 Higher efficiency (>110%)

 If 10-fold dilutions are all >3.32 cycles apart:

 The concentration of inhibitors is maximum in the least dilute

Master Mix B Master Mix B

Replicates ideally should not be

 Lowered background due to improved quenching

 Calibrate the machine

 Amplification is observed beyond plateau

 Information on PrimeTime® qPCR Assays with ZEN™ double-quenched probes and

 For background on setting up qPCR experiments, qPCR protocols, and troubleshooting

 Information on products that can be used as controls such as MiniGenes™, gBlocks™,

Least ROX ROX Normalization =OFF

 Differences in ROX concentration

You might also like