Despite these standardized abbreviations, it is important to note that this nomenclature guideline is not always adhered to, and qPCR is commonly used to describe RT-qPCR. This is a technique which combines RT-PCR with qPCR to enable the measurement of RNA levels through the use of cDNA in a qPCR reaction, thus allowing rapid detection of gene expression changes (see Figure 1C). The final acronym ‘RT-qPCR’ is used for reverse transcription quantitative real-time PCR. This technique is used to detect the presence of pathogens and to determine the copy number of DNA sequences of interest. According to MIQE, the acronym ‘qPCR’ describes quantitative real-time PCR, which is the PCR amplification of DNA in real time, measured by a fluorescent probe, most commonly an intercalating dye or a hydrolysis-based probe, enabling quantitation of the PCR product (see Figure 1B). This technique can be used in molecular cloning of genes of interest (GOIs), but most commonly, it serves as the first step in RT-qPCR. This can then be amplified by a DNA polymerase, generating double-stranded cDNA, feeding into a standard PCR-based amplification process (see Figure 1A).
Using the reverse transcriptase enzyme, a single-stranded copy of cDNA is generated. Reverse transcription PCR allows the use of RNA as a template to generate complementary DNA (cDNA). They stated that ‘RT-PCR’ should only be used to describe reverse transcription PCR and not real-time PCR, as is often confused. In an attempt to prevent this, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, first published in 2009, proposed a standardization of abbreviations. Indeed, the similarities between the closely related techniques often result in the incorrect use of the acronyms. When discussing this topic, it is important to highlight the common misconception that RT-PCR, qPCR and RT-qPCR are synonymous.
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