Let’s Talk about Real-time PCR! Part 2

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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 PCR (qPCR) reactions?  Here are some commonly experienced issues, and some possible solutions to try.

 

1. 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 a lower efficiency, including inefficient annealing between the primers and target, the primers binding to competing sites, the 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.

 

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

A PCR efficiency greater than 1 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. You may need to dilute the sample to dilute out any contaminants.

 

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

Your samples may have evaporated if they were not stored correctly, increasing the concentration of the target. 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.

 

4. Why is there amplification in the no template control?

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. Find more qPCR tips and solutions in the free Azure qPCR Troubleshooting guide.

Let’s talk about real time PCR! Part 1

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qPCR

What is qPCR and how is it used in practice? Quantitative real-time PCR 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. PCR transformed biological research, and now, during the COVID pandemic, more people are talking about PCR than ever before.

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 will be a two-part blog series. In Part I, let’s define some terms:

PCR: Polymerase chain reaction. The development of this technique to amplify specific pieces of DNA won its inventor the 1993 Nobel Prize in chemistry. PCR takes advantage of the double-strandedness of DNA. The DNA in a sample is sequentially heated to separate the strands, cooled to allow 2 primers, each specifically designed to bind to one strand bracketing the sequence to be amplified, to anneal to the separated DNA strands, and then 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 is amplifying 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, to construct recombinant plasmids, to use as probes in new experiments, to use in a diagnostic test, or other downstream applications.

RT-PCR: RT-PCR is “reverse transcription PCR”.  What if you want to detect or quantify RNA, which is single stranded?. The answer is RT-PCR; RNA is reverse transcribed (made into DNA) and then that DNA is subjected to PCR. Luckily, almost all messenger RNAs (mRNAs) that encode proteins have poly-A tails. So, by using a poly-T primer, all messenger RNAs can be reverse-transcribed into their complementary DNA sequence (cDNA).

Real-time PCR: In real time PCR, the accumulation of PCR products is followed in real time by incorporating fluorophores into the product and monitoring the fluorescence of the reaction.  There can be confusion around the term RT-PCR; convention is that RT-PCR stands for reverse transcription PCR and not real time PCR.

Ct value: The Ct value is the “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: qPCR is quantitative PCR. Real time PCR 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 future blogs, we’ll discuss PCR applications and troubleshooting.

Learn more about qPCR applications and Azure’s Real-time PCR instrument, the Cielo by clicking here.