Two-Step RT-PCR

Reverse transcriptase with RNase inhibitor and buffer. The following viral reverse transcriptases are the most common used enzymes for the cDNA synthesis in two-step RT-PCR  

Using the two above-mentioned primer systems, all cellular mRNA is transcribed to cDNA, which gives the advantage to amplify different targets in addition to positive controls simultaneously, also as a multiplex PCR. 

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To analyze RNA by PCR an additional step has to be introduced to transcribe RNA into cDNA, which is a more stable single-strand DNA complementary to the targeted RNA. This can be achieved by the reverse transcription (RT) of the template RNA using a reverse transcriptase enzyme. RT and PCR can be carried out either sequentially in the same tube (one-step RT-PCR) or separately (two-step RT-PCR). One-step RT-PCR requires gene-specific primers for the reverse transcription reaction, whereas in two-step RT-PCR random primers can be used. 

Reverse transcriptase (RT) PCR (RT-PCR) can be used to detect RNA in specimens, especially ssRNA viruses. Using the ssRNA as a template, cDNA (complementary DNA) can be synthesized with the enzyme reverse transcriptase, which can further be used as a template in PCR amplification. In two-step RT-PCR, extracted RNAs are first mixed with RT and suitable primer to synthesize cDNA and then followed by adding Taq polymerase and PCR primer pairs for PCR amplification. In one-step RT-PCR, all the ingredients are mixed together and allowed for one cycle of reverse transcription to synthesize cDNA followed by 30 + cycles of PCR amplification. The product can be analyzed by agarose gel electrophoresis. Commercial RT-PCR test kits are available. 

Reverse transcription and PCR amplification can be performed as a two-step process in a single tube or with two separate reactions. RT-PCR performed on fresh-frozen tissue provides high-quality amplification and reliable results. However, when FFPE tissue is used for RT-PCR analysis, the results vary and depend on the level of RNA degradation and length of PCR amplification. To attain a more stable RT-PCR amplification from FFPE tissues, it is typical to choose a target that is less than 150 to 200 nt long. 

Extraction of DNA from biological samples can be accomplished by precipitation or affinity methods [15]. Amplification of the amount of DNA is also needed before any sequence detection can be done. This can be done by a method known as polymerase chain reaction or PCR in short. This process is depicted in Figure 8.6. Briefly, original double stranded DNA molecules, shown as black rectangles, are heated to more than 90°C for separation. Afterward, DNA primers, shown as squares, as well as nucleic acids are added to the solution to initiate the DNA synthesis, forming two pairs of double stranded DNA at the end of the first cycle. The process is repeated for more than 30 times, doubling the amount of DNA at each step [16]. RNA can also be amplified using a similar process known as reverse transcription-polymerase chain reaction (RT-PCR) [17]. 

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