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

Diagram showing the difference between one-step and 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-PCRTwo-step RT-PCR
SetupCombined reaction under conditions that support both reverse transcription and PCRSeparate optimized reactions for reverse transcription and PCR
PrimersGene-specific primersChoice of oligo(dT), random hexamers, or gene-specific primers
Ideal useAnalysis of a few genes; high-throughput platformsAnalysis of multiple genes
Advantages
  • Less experimental variation since both reactions take place in the same tube
  • Fewer pipetting steps helps reduce risk of contamination
  • Suitable for high throughput amplification/screening 
  • Fast and highly reproducible
  • A stable cDNA pool is generated that can be stored for long periods of time and used for multiple reactions
  • The target and reference genes can be amplified from the same cDNA pool without multiplexing
  • Optimized reaction buffers and reaction conditions can be used for each individual reaction
  • Flexible priming options
Disadvantages
  • Impossible to optimize the two reactions separately
  • Less sensitive than two-step because the reaction conditions are a compromise between the two combined reactions
  • Detection of fewer targets per sample
  • The use of several tubes and pipetting steps exposes the reaction to a greater risk of DNA contamination
  • Time consuming
  • Requires more optimization than one-step

Learn more: Reverse transcription products

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