5. 5
R, Rn and Delta Rn
R= Multicomponent view (fluorescence obtained without any normalization)
Rn: Normalized reporter signal = emission intensity of the reporter dye
emission intensity of the passive reference dye (ROX)
ΔRn = Rn – background fluorescence
5
6. 6
Linear Rn View Log Baselined dRn
Baseline stop value should be set 1 to 2 cycles before earliest amplification cycle
Baseline should be set in the linear view
Improper Baseline
6
7. 7
Proper Baseline
Linear View Log View
Baseline stop value should be set 1 to 2 cycles before earliest amplification cycle
Baseline should be set in the linear view
8. 8
Good Threshold –
in exponential phase
Bad Threshold –
in plateau phase
Bad Threshold –
in baseline phase
Threshold
Linear Scale
Logarithmic Scale
Linear view for Baseline setting
Log view for Threshold setting
8
10. 10
No Amplification
Lack of target in sample
Positive control
Assay design failure
Try a different assay
Sample degradation
Does a different cDNA prep give
you the same result?
Machine not calibrated for dye being used
Calibrate the instrument
Incorrectly assigned dye detector
Make sure setting on instrument matches
the probe being used
Log
Linear
FAM assigned as TAMRA
FAM assigned as TET
12. 12
PCR Efficiency
Lower efficiency
Primers designed on a SNP site
Lower sensitivity of probe
Sample inhibition
Incorrect dilutions causing errors in standard curve
Higher efficiency (greater than 110%)
Primer dimers or nonspecific amplification
Incomplete DNase treatment
Incorrect dilutions causing errors in standard curve
Not enough dynamic range of standard curve
15. The shift due to a SNP at the
3′ end of a primer varies
from 0 to >10 Cq’s.
This shift misrepresents a
gene expression fold change
of as much 1000 fold
Impact of SNPs on Primer Efficiency
Effect of SNPs within primer locations on Tm
16. PrimeTime® Predesigned qPCR Assays for Human, Mouse, and Rat
• Designed to avoid SNPS
• We share primer and probe sequences upon purchase
17. 18
Delayed Cq……Sample Inhibition
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 contaminantion with phenol layer
during RNA isolation
10 fold dilution
19. 20
Delayed Cq……..Lower Efficiency
If 10 fold dilutions are all greater than 3.32 cycles apart…
Are your primers on a SNP site?
Can a difference in primer Tms (> 5 °C) be producing unequal extension
Annealing temperature is too low
Unanticipated variants within the target sequence
21. 22
Early Cq…..Too Much Template
Too much template
Cq value comes up before 15
True amplification is observed when analyzed in the linear view
22. 23
Early Cq…..Automatic Baseline Failure
When too much template is present, it is likely that the software is
unable to distinguish between noise and true amplification, thus
auto baseline may incorrectly assign the value for the baseline
correction factor
Adjusting baseline manually corrects this problem
23. 24
Earlier than expected Cq
Genomic DNA contamination
Multiple products
High primer-dimer production
Poor primer specificity
Transcript naturally has high expression in samples of interest
24. 25
Scattered Replicates
Pipetting Errors
Poor thermal calibration (inconsistent raising and lowering of
temperature across different wells in a thermocycler block)
Denaturation time is too short ( if using fast cycling master mix
(consider increasing denaturation time from 5 to 20 secs)
Low copy number
Incorrectly set baseline
26. 27
Height of Amplification Curve
Lowered background
Probe concentration
Signal bleed over
Incorrectly assigned detector
Increased ROX in samples
Master mix
27. 28
Lowered background due to improved quenching
IDT ZEN™ Double-Quenched Probes (available with IDT PrimeTime® qPCR Assays) have lower
background and increased sensitivity
ZEN™ Double-Quenched Probes
Height of Amplification probes…Lowered Background
28. 29
Regular qPCR Dual-Labeled Probes
ZEN™ Double-Quenched Probes
Dyes FAM, TET, HEX™, MAX, or JOE
Internal Quencher ZEN™
3′ quencher Iowa Black® FQ
FAM/ZEN/IaBlkFq is available as:
• PrimeTime® Mini Probes—0.5 nmole delivered yield
• PrimeTime® Eco Probes—2.5 nmole delivered yield
Also available on starting synthesis scales of 100 nmole, 250 nmole
and 1 µmole
PrimeTime® qPCR—ZEN™ Double-Quenched Probes
29. Case Study—How ZEN™ DQP Makes the Difference
Adding a ZEN™ Internal Quencher decreases background fluorescence
Figure 1A). Railing can lead to signal bleed over into adjacent channels, which can complicate data interpretation if those channels are
also being used (Figure 1B). The reduced background fluorescence of ZEN™ Double-Quenched Probes compared to traditional single-
quenched probes is demonstrated in Figure 1C.
30. 31
Height of Amplification Curve……Incorrect Probe Concentration
Correct Probe
Concentration
Incorrect Probe
Concentration
Lowered height of amplification curve can also be
due to limiting reagents or degraded reagents
such as the dNTPs or master mix
32. 33
Height of Amplification cCurve….Low ROX
Normalized reporter signal (Rn):
emission intensity of the reporter
emission intensity of the passive
reference dye (Rox)
ΔRn = Rn – background
noise
50 nM ROX
100 nM ROX
33. 34
Height of Amplification Curve……Multiplex vs Singleplex
Height of amplification curves are typically lowered when a target is
investigated in a multiplex reaction in comparison to a singleplex reaction
More importantly though it is important that the Cq is not shifted between
both reactions
If multiplexing, master mix needs to be adjusted for additional
dNTPs, Mg2+ and Taq enzyme or use a master mix specifically designed
for multiplexing
Singleplex
Multiplex
40. 41
Unusual Curve……..Negative Curves
If the instrument is not correctly calibrated, when fluorescence due to
amplification increases in a given channel, the fluorescence attributed to
background increases, while fluorescence attributed to the other dyes may
be decreased by the instrument
Calibrate the machine again for all the dyes being used
41. 42
Unusual Curves….Amplification Beyond Plateau
Amplification is observed beyond plateau
Fluorescence detected is at maximum capacity for the detector
The amount of fluorescence attributed to ROX is mistakenly
decreased as the amount of fluorescence attributed to back ground
increases
Fluorescence is normalized
to a smaller Rox value,
artificially increasing the
height of the amp curve
Turn normalizer off
Lower primer probe conc.
45. 46
Melt Curves, An Indicator, Not a Diagnosis
(A) An amplicon from CFTR
exon 17b reveals a single
peak following melt curve
analysis, while
(B) An amplicon from exon 7
produces 2 peaks, often
considered as
representing multiple
amplicons.
46. It Takes More Than a Melt Curve
C. uMelt Derivation Melt Curve
for CFTR Exon 13.
B. CFTR Exon 13
Agarose Gel.
CFTR Exon 13 Melt Curve.
Shoulder peaks maybe due to low complexity regions in your amplicon that cause
non-uniform melting
Typically, primer-dimers have a significantly lower melting temperature and present
with a low, broad melting curve peak.
47. qPCR Resources: Webinars & Technical Info
For More information please visit www.idtdna.com Support Tech & Ed. Materials
48. 49
PrimeTime® qPCR Products
Gene Expression Studies
Custom design in any species
ZEN™ Double-Quenched Probes
In human, mouse, and rat
PrimeTime® qPCR Predesigned qPCR Assay Database
Genotyping Studies
Custom design in any species
LNA PrimeTime® Probes and Mini LNA PrimeTime® Probes
Free Design Tools
Custom design in any species
PrimerQuest ® Tool, RealTime PCR Tool
Resources on the Web