Reverse transcription polymerase chain reaction (RT-PCR)

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA.

This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.

The close association between RT-PCR and qPCR has led to metonymic use of the term qPCR to mean RT-PCR. Such use may be confusing,[2] as RT-PCR can be used without qPCR, for example to enable molecular cloning, sequencing or simple detection of RNA. Conversely, qPCR may be used without RT-PCR, for example to quantify the copy number of a specific piece of DNA. PCR (polymerase chain reaction) tests are a fast, highly accurate way to diagnose certain infectious diseases and genetic changes.

The tests work by finding the DNA or RNA of a pathogen (disease-causing organism) or abnormal cells in a sample. What is real time RT–PCR? Real time RT–PCR is a nuclear-derived method for detecting the presence of specific genetic material in any pathogen, including a virus. Originally, the method used radioactive isotope markers to detect targeted genetic materials, but subsequent refining has led to the replacement of isotopic labelling with special markers, most frequently fluorescent dyes.

This technique allows scientists to see the results almost immediately while the process is still ongoing, whereas conventional RT–PCR only provides results at the end of the process. Applications Of RT-PCR Real time RT–PCR is one of the most widely used laboratory methods for detecting the COVID-19 virus. While many countries have used real time RT–PCR for diagnosing other diseases, such as Ebola virus and Zika virus, many need support in adapting this method for the COVID-19 virus, as well as in increasing their national testing capacities. RT-PCR: A Science and an Art Form RT-PCR is that technology by which RNA molecules are converted into their complementary DNA (cDNA) sequences by reverse transcriptases, followed by the amplification of the newly synthesized cDNA by standard PCR procedures.

This approach to study gene expression is universally known as RT-PCR, because of the role of reverse transcriptase (RT) in the synthesis of first-strand cDNA. RT-PCR is a two-step process. It involves reverse transcription of purified RNA by RT via an appropriate method for priming and amplification of first-strand cDNA using some variant of PCR.

Normalization of samples is very important in RT-PCR, and the efficiency of first-strand cDNA synthesis is one of the most important determinants in the success or failure of this method. For this reason, it is strategically better to make a large cDNA pool from which aliquots may be drawn for individual applications rather than repeating the same cDNA synthesis reaction over and over.

Designing useful primers necessitates promoting a proper balance between template specificity, thermodynamic stability when base-paired to the template, and capacity of one primer to function with the other(s) to support RT-PCR. The probable collaborative behavior of one or more pairs of oligonucleotide primers is best described in terms of the Tm of each primer involved. The Tm is that temperature at which 50% of the possible annealing events between primer and template have occurred and 50% have yet to occur.

Gaurav Singh

Editor in Chief Medical Microbiology & RDT Labs - RDT Labs Magazine

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