Let’s Talk about qPCR! Part 2

qPCR Troubleshooting

This is part two of our “Let’s Talk about Real-time PCR” post, so check out part one if you haven’t yet.

Are you seeing strange or unexpected results in your quantitative real-time PCR (qPCR) reactions?  Here are some commonly experienced issues, and some possible solutions to try.

Why is my PCR efficiency so low?

The efficiency of a PCR reaction is the fraction of target molecules copied every PCR cycle. In general, an efficiency of at least 90% is recommended. Many factors can contribute to low PCR efficiency, including

  • Inefficient annealing between the primers and target,
  • The primers binding to competing sites,
  • Presence of inhibitors in the sample, and
  • Insufficient reagents in the mix.

Double check the primer sequences to make sure there are no potential competing reactions. Run a melting curve to make sure you aren’t amplifying unexpected products. Reaction conditions may need to be optimized.

My PCR efficiency is too high! How is it possible to have an efficiency greater than 1?

If your PCR efficiency is high, or greater than 1, this would suggest that more than 100% of the targets are replicated each cycle. What’s going on? The presence of inhibitors in the sample is frequently the source of efficiency measures greater than 1. The greatest inhibition is in the most concentrated samples used in a dilution series, so the effect of inhibition on the standard curve is more pronounced at one end and distorts the slope of the curve, changing the efficiency calculation. To fix high PCR efficiency, you may need to dilute the sample to dilute out any contaminants.

Why are my Ct values so low? There's no way there was that much target in my sample.

Your Ct values are low because your samples may have evaporated if they were not stored correctly, which increases the concentration of the target. To fix this, carry out a melting curve at the end of PCR to make sure you are only amplifying the expected target and not amplifying something unexpected or primer dimers.

Why is there amplification in the no-template control?

A reason there is amplification in the no-template control could be due to contamination. Contamination is a major consideration when carrying out PCR. Make sure your pipettes and workplace are clean so you are not potentially transferring amplified products from a previous experiment into your solutions. Also, run a melting curve to see if you are amplifying primer dimers.

We hope this is helpful as you troubleshoot your qPCR. You can find more qPCR tips and solutions by downloading our free qPCR Troubleshooting guide.

Let’s talk about qPCR! Part 1


What is qPCR and how is it used in practice?

PCR (polymerase chain reaction) transformed biological research. During the COVID pandemic, more people were talking about PCR than ever before. Quantitative real-time PCR (qPCR) is a powerful technique for showing not only whether a nucleic acid sequence is or is not present in a sample, but for determining exactly how many copies of that sequence are present in the sample.

So what is it? How is it used? And where do you turn when your reactions aren’t working how you expected them to? This is part I of a two-part blog series. In Part I, let’s define some terms:

What is PCR: Polymerase chain reaction

The development of the PCR technique to amplify specific pieces of DNA won its inventor, Kary B. Mullis, the 1993 Nobel Prize in Chemistry. PCR takes advantage of the double-strandedness of DNA. The steps in PCR are: 1) DNA in a sample is sequentially heated to separate the strands, 2) cooled to allow two primers (each specifically designed to bind to one strand bracketing the sequence to be amplified, to anneal to the separated DNA strands) and 3) incubated with a DNA polymerase which binds to the annealed primers and synthesizes the complementary DNA strands.

At the end of each cycle, one piece of double-stranded DNA has been copied into two double-stranded pieces. Over subsequent PCR cycles, each copy is itself copied, so the number of copies grows exponentially.

Because PCR amplifies the target sequence exponentially, if you start with a single piece of DNA, after 20 cycles of PCR, there will be over a million copies of the target sequence. PCR can be used to make enough copies to visualize on a gel, construct recombinant plasmids, use as probes in new experiments, use in a diagnostic test, or in other downstream applications.

What is qPCR: Real-time PCR

Let’s clear the air. There can be confusion around the term RT-PCR, which stands for “reverse transcription PCR.” Real-time PCR is commonly known as qPCR (the “q” stands for quantitative”), where the accumulation of PCR products is followed in “real time” by incorporating fluorophores into the product and monitoring the fluorescence of the reaction.

Ct value: cycle threshold value

When watching fluorescence increase as PCR products accumulate in real time, initially the fluorescence signal will be too low for the detector to pick up. Scientists select a threshold value above which the fluorescence signal can be confidently detected. To build a standard curve, they see how many cycles it takes for a reaction with a known starting amount of target sequence to reach the fluorescence threshold value. That cycle number is Ct.

qPCR: quantitative real-time PCR

qPCR allows quantitation of the amount of target in a sample. Comparing the increase in fluorescence signal of the sample to a standard curve made by amplifying known starting amounts of the target sequence, the amount of target in an unknown can be calculated.

In the part II of this series, I’ll discuss PCR applications and how to troubleshoot them.

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