RT-PCR can be performed in a one-step or a two-step assay. One-step assays combine reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. One-step RT-PCR only utilizes sequence-specific primers. In two-step assays, the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies.
Take a look: One-step vs two-step RT-PCR
Figure 1. One-step vs. two-step RT-qPCR.
What is one-step RT-PCR?
As the name implies, one-step RT-PCR combines first-strand cDNA synthesis (RT) and subsequent PCR in a single reaction tube. This reaction setup can help simplify your workflow, reduces variation, and minimizes possible contamination. One-step RT-PCR allows easy processing of large numbers of samples, making it amenable to high-throughput applications. However, one-step RT-PCR uses gene-specific 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 could be less sensitive and less efficient in some scenarios. Nevertheless, use of a gene-specific primer in RT-PCR can help maximize the yield of the target cDNA and minimize background amplification.
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What is two-step RT-PCR?
Two-step RT-PCR entails two separate reactions, beginning with first-strand cDNA synthesis (RT), followed by amplification of a portion 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 RT and PCR reactions allows for optimization of reaction conditions for each step, as well as flexibility with reverse transcription priming (oligo(dT) primers, random hexamers, or gene-specific primers) and PCR setup (e.g., DNA polymerase choice and PCR components). Compared to one-step RT-PCR, the disadvantages of two-step RT-PCR include multiple steps for an extended workflow, additional sample handling and processing, and may increase the chance of contamination and variation in results.
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Table 1. Comparison of one-step vs two-step RT-PCR
| One-step RT-PCR | Two-step RT-PCR | |
|---|---|---|
| Setup | Combined reaction under conditions that support both reverse transcription and PCR | Separate optimized reactions for reverse transcription and PCR |
| Primers | Gene-specific primers | Choice of oligo(dT), random hexamers, or gene-specific primers |
| Ideal use | Analysis of a few genes; high-throughput platforms | Analysis of multiple genes |
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Learn more: Reverse transcription products