“The question I am asked a lot is, am I doing my real-time PCR reactions the right way?” Marcia Slater started working on real-time PCR (also known as qPCR) 20 years ago, at the very start of the development of the method. She has gained a wealth of information from her experience working on this technique, both as a Field Applications Specialist and now in marketing.
The polymerase chain reaction (PCR) is one of the most powerful technologies in molecular biology. Using PCR, specific sequences within a DNA or complementary DNA (cDNA) template can be copied, or “amplified,” many thousand- to million-fold using sequence-specific oligonucleotides, heat-stable DNA polymerase and thermal cycling. In real-time PCR, also often called qPCR, we measure the amount of DNA (the amplicon) each cycle using fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product molecules (amplicon) generated. Ideally, with each PCR cycle, every target DNA molecule will be doubled. We collect data throughout the cycling and then focus the analysis on the exponential phase of the reaction, where this doubling takes place. That is how we get information on the starting quantity of the DNA target using fluorescent reporters. The change in fluorescence is constantly measured by a thermocycler with fluorescent dye scanning capacity. By plotting fluorescence against the cycle number, the real-time PCR instrument generates a graph that represents the accumulation of product over the duration of the entire PCR run. It means that you can see what happens during the reaction, which gives you a lot of additional information about the real-time PCR experiment.
qPCR is More Complex Than Perceived
qPCR can be really simple to do, but it can also be really hard, says Slater. According to a recent publication, qPCR is more complex than perceived by many scientists—especially the qPCR experiments used in scientific research settings, where all primers and probes need to be designed for a specific detection. Experiments often involve the quantification of nucleic acid levels between two different experimental test circumstances. “When the whole of your future research is based on these results, you really want to get it right,” laughs Slater. “In my experience, inadequate and different protocols between laboratories have also been a real frustration for reproducing data.”
Minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines have helped the scientific community produce more reliable and consistent data. But concerns remain about the quality of published qPCR data.
“I find that once you dig into the background of qPCR a little deeper and take the time to study the different critical steps in qPCR experiments, it suddenly gets much easier,” Slater says.
Some of the most critical aspects of setting up qPCR experiments are the choice of primers and probes, the best controls to use in your experiments, and melting curve analysis if you are using SYBR Green.
The qPCR handbook worth checking out
Slater’s advice is to check out the new qPCR handbook from Thermo Fisher Scientific. It consists of interactive modules that walk you through all the critical steps of how to set up a qPCR experiment. It is a great resource, whether you are new to qPCR or you want to refresh your knowledge and check whether your reactions are still optimal. The first module is now available. It gives you an introduction to real-time PCR and takes you all the way to all the different forms of PCR and their applications, right through to how to choose internal controls and instrument calibration.
For research use only. Not for use in diagnostic procedures.