TransGen Biotech

For RT-PCR applications, there are two approaches: one-step RT-PCR and two-step RT-PCR. Each of these methods has its advantages and disadvantages. As its name implies, single-step RT-PCR combines the synthesis of complementary DNA (cDNA) of the first strand (reverse transcript, RT) and subsequent PCR in a single reaction tube. This reaction configuration simplifies the work, reduces variations and minimizes possible contamination. One-step RT-PCR makes it easier to process a large number of samples, allowing for high-throughput applications. However, one-step RT-PCR uses specific gene primers for amplification, limiting the analysis to a few genes per RNA sample. Since the reaction is a compromise between reverse transcription and amplification conditions, one-step RT-PCR may be less sensitive and less effective in some cases. However, the use of a gene-specific primer in RT-PCR can help maximize the yield of the target cDNA and minimize amplification.

The two-step RT-PCR involves two distinct reactions, starting with first strand cDNA synthesis (reverse transcription-RT) and then amplifying part of the resulting cDNA by PCR in a separate tube . Therefore, two-step RT-PCR is useful for detecting multiple genes in a single RNA sample. The separation of the RT and PCR reactions makes it possible to optimize the reaction conditions for each step as well as the flexibility with priming by reverse transcription (oligo (dT) primers, random hexamers or gene-specific primers) and PCR DNA polymerase and PCR components). Compared to one-step RT-PCR, the disadvantages of two-stage RT-PCR include several steps for extended workflow, additional handling and processing of the sample, and increase the risk of contamination and variation results.