Steps of RT-PCR Technique As mentioned above, the RT-PCR process is only a little different from that of PCR. Apart from the one addition of reverse transcription in RT-PCR, both techniques are almost the same. So, let’s start the RT-PCR technique from its reverse transcription stage. Reverse Transcription A reverse transcriptase enzyme is used to synthesize a cDNA (complementary DNA) strand with nucleotides, and its end product turns out to be mRNA (cDNA hybrid). This process takes place between 40 to 50 degrees Celsius; it usually varies with the properties of the reverse transcriptase enzyme being used. Once the reverse transcription is done, the mRNAs are hydrolyzed, and during a first temperature cycle, the cDNAs are replicated by DNA polymerase. Denaturation Here, the combination of components left is heated to a temperature of 94 degrees Celsius for less than half a minute. Along with the denaturation of double-stranded cDNA into single strands, the hydrogen bonds are also...
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