In case the name doesn't give it away, real-time PCR is a PCR application that monitors DNA amplification in real time. This means that amplification is monitored during the PCR reaction, and not at the end of the reaction as with end-point PCR, where PCR products are typically analyzed post-run on agarose gels.
This article is the first in a two-part series. In part 1, we will go through the basics of real-time PCR, including its advantages over end-point PCR, the workflow, the typical data output, the choice of fluorescent labeling systems available and the pros and cons of each, while part 2 will cover the different quantification methods available, setup tips, primer design and quality control.
Real-Time PCR vs. qPCR vs. qRT-PCR, and so on...
Because DNA amplification is monitored in real time in a specialized PCR instrument, real-time PCR is a quantitative method, and for this reason it is also commonly referred to as qPCR (quantitative PCR). When real-time PCR is used to monitor gene expression by quantitative analysis of cDNA (i.e., reverse-transcribed RNA), it may be referred to as real-time RT-PCR, qRT-PCR, or modifications of these. We will use 'real-time PCR' throughout this article to avoid confusion.
For standard guidelines on real-time PCR nomenclature and how to report results, check out the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) (1).
Real-Time PCR Workflow
The real-time PCR workflow is quite straightforward, and the PCR reaction setup is similar to end-point PCR.