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Overview of Reverse Transcription Real-Time Quantitative Polymerase Chain Reaction

Sonal Priya
Sonal Priya
Graduate Student, Department of Chemistry
University of Georgia
ONLINE ONLY
Analytical Seminar

Polymerase Chain Reaction (PCR) is an in vitro enzymatic method used to amplify specific DNA sequences. The simple concept of PCR relies upon the repeated synthesis of targeted DNA using DNA polymerase enzyme. Conceived by Kary Mullis in 1983, PCR has now become a common and often indispensable technique that is used in clinical laboratory and medical laboratory research for a broad variety of applications including biomedical research and criminal forensics. Reverse Transcription quantitative PCR (RT-qPCR) is a modification to polymerase chain reaction that allows precise quantification of specific nucleic acids in a complex mixture by fluorescent detection of labeled PCR products. While this technique has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is essential. The RNA is reverse transcribed into complementary DNA (cDNA) using reverse transcriptase prior to standard PCR, which proceeds with template denaturation, primer annealing and primer extension steps. Two general methods for quantitative detection of the amplicon include non-sequence specific fluorescent intercalating dsDNA binding dye (SYBR Green I), and gene-specific fluorescent probes based on fluorescence resonance energy transfer (FRET). The best-known gene-specific probe-based system is TaqMan, which makes use of 5'-3' exonuclease activity of Taq polymerase to quantitate target sequences in a sample. A successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. By using negative and positive control, one can monitor any contamination introduced during laboratory testing and ensure proper functioning of amplification reagents and equipment respectively. In addition, melting point analysis feature can be utilized to distinguish target amplicons from PCR artifacts such as primer-dimer or mis-primed products. The amount of initial target RNA, and in some cases, the levels of expressed genes may be measured by absolute or relative quantification. Furthermore, to face the on-going COVID-19 pandemic, RT-qPCR is considered as the gold standard for detection of genomic viral RNA with high sensitivity and specificity.

 

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[3] VanGuilder, H. D.; Vrana, K. E.; Freeman, W. M. Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques 2008, 44, 619.

[4] Kuang, J.; Yan, X.; Bishop, D. J. An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research. PLOS ONE 2018, 13, 5, 1964.

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