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.