qPCR Flashcards
What is “absolute” quantification?
Claims to determine the exact number of copies present
Why is absolute quantification more difficult for RNA/cDNA?
Difficult to account for variation during cDNA synthesis.
What is “relative” quantification?
Quantifification of target relative to a control, often an internal reference gene or externally spiked template (SPC)
What is the main assumption of the ddCt method?
Efficiencies of reference and target are equal
Why is the Pfaffl method sometimes considered advantageous compared to the ddCt method?
It takes into account the efficiencies of the calibrator and the target
Are the dCt and ddCt methods the same?
No, the dCt method is based only on the standard curve. The ddCt method requires an external calibrator.
How is qPCR analyzed in our lab?
Use ddCt method w/ an external calibrator AND a standard curve for the target to get fully quantitative results; only accept a narrow range of efficiencies
SPC
specimen processing control
What are the main components of a std curve?
slope
y intercept
r2
What is an acceptable r2 value?
> 0.995
What does the slope of a standard curve tell you and what are acceptable values?
slope is an indication of efficiency; want between -3.1% and -3.58% (90-110%)
What slope is equal to 100% efficiency?
-3.32
What is the optimal amplicon length for qPCR?
75-200 bp
shorter amplicons amplify more efficiently
Why do amplicons need to be at least 75 bp?
so that you can distinguish them from primer dimers
Why is it especially important to avoid secondary structure at the 3’ end of a primer or probe?
Because the DNA polymerase can attach and extend the primer creating primer dimers
Why should a GC clamp be added to the 3’ end of a primer?
Helps promote specific binding at the 3’ end (GC bond is stronger than an AT bond). This increases PCR efficiency.
What %GC should primers be? Why?
50-60%; GC bonds are stronger, this promotes stable primer/probe binding
True or false: primers do not need to have roughy the same %GC
false
True or false: primers need to have the same annealing temp
true (within 5degC)
Why should runs of 3 or more GCs be avoided when designing primers
May promote mispriming because of stability of annealing
True or false: you should avoid >4bp repeats in a PCR primer or probe.
true
Why is it important to verify primer and probe sequences with BLAST?
To ensure that your assay is not cross-reactive for other sequences (will result in false positives)
How much higher should the Tm of the probe be than the primers? Why?
5-10 degC
If the melting temperature is too low, the percent of probe annealed will be lower than the percent of primers; even though primers are being extended during the reaction, the assay will lack sensitivity due as no fluorescence will be detected for that duplication event.
What %GC should probes have?
30-80%
How long should primers be?
15-25 bp (~20 bp is optimal)
How long should probes be?
20-30 bp
Why is important that probe sequences don’t have a G at the 5’ end?
Can quench fluorescence of the 5’ fluorophore
What Tm is optimal for a probe?
5-10 degC higher than the primers
True or false: probes should be designed to anneal to the strand with more Cs
false; probes should be designed to anneal to the strand with more Gs
why is it important to run a melt curve for a Sybr Green assay?
To ensure that products have the same/expected melting temperature
Why are Taqman assays considered more specific?
Because they require the binding of primer AND probe sequences
How do you optimize annealing temperature for an assay?
Run a gradient block, choose highest temperature that yields good efficiencey (between 95-105% eff.)
How do you optimize primer/probe concentration for an assay?
Run various concentrations with various primer/probe concentrations. Want the concentration that yields the best amplification and the lowest background fluorescence
What is a good starting annealing temperature for a qPCR assay?
3-5 deg lower than primer Tm, should be kept as high as possible to preserve the specificity of the reaction
Water are the typical components of qPCR mastermix?
water, buffer, Mg++ or K+, dNTPs, ROX ref (optional), Taq DNA polymerase
Add primers, probes, template for reaction to proceed
Which component of mastermix can be adjusted for better specificity?
Mg++, a cofactor for the DNA polymerase
What happens to the reaction when more Mg++ is added? Less?
Mg++ concentrations control the specificity of the PCR reaction. More Mg++ causes more Taq activity but less specificity. Less Mg++ causes less Taq activity and more specificity
Pick one: Most PCR inhibitors are A: Organic or B: Inorganic.
Organic
What are examples of PCR inhibitors in oysters?
polysaccharides, glycogen
Make it difficult to resuspend DNA and can “mimic” DNA, disrupting the enzymatic process
What are some common PCR inhibitors in water?
humics, tannic acid, ions (Ca2+), other debris
metal ions can competitively bind the polymerase, humics and tanic acid interact with both template and polymerase even at very low concentrations
What is the Monte Carlo Effect? What causes it?
“An inherent limitation of QPCR due to small amounts of complex template”
“Caused by differences in amplification efficiency between individual template molecules.”
“The lower the template abundance, the less likely its true abundance will be reflected in the amplification.”
Possibly caused by a limiting number of template molecules– if primers don’t anneal properly due to decreased template concentration then that template will have lower efficiency
When is the Monte Carlo Effect most likely to occur?
In the beginning of a rxn, can cause large variation in the final template numbers
What is one way to avoid the Monte Carlo Effect?
Test assay sensitivity with a standard curve, the highest dilution of the standard curve that results in consistent Ct values is the concentration of target that can be quantified with confidence
What is the LOB?
Limit of blank; defined as the 95th percentile of all Cts for blanks
How is the LOB calculated?
- run at least 10 blanks
- find 95th percentile (5th for Cts in Excel)
- Use that Ct value to calculate LOB in copies for the assay
What is the max acceptable Ct difference between replicates?
0.5
What is the plateau effect?
Nonspecific products formed during the beginning of the PCR reaction begin to accumulate, causing the actual yield of the reaction to plateau
How should you choose the best primer/probe concentration? (3 items)
Lowest background fluorescence
Earliest Ct value
Greatest sensitivity and specificity
