What’s the difference between one-step and two-step RT-PCR?

One-step RT-PCR reactions are easier to set-up, shorter on time, and use less reagents. So, why aren’t they used exclusively in the lab?  Let’s start by defining what RT-PCR is used for:

What is RT-PCR?

PCR (polymerase chain reaction) is a technique used for amplification of DNA strands. Discovered by Kary B. Mullis in the early eighties, this approach is typically considered the gold standard in the analysis of nucleic acid. When working with RNA as a starting material, however, RT-PCR (reverse transcription-PCR) is used to obtain DNA from the RNA template. This is done by converting RNA into cDNA (complementary DNA) using RT-PCR, and then amplifying the cDNA into DNA via standard PCR.

How is RT-PCR done?

RT-PCR is accomplished using a reverse transcriptase enzyme such as Invitrogen SuperScript IV. Reverse transcription can be accomplished using two approaches: one-step or two-step RT-PCR.

What is one-step RT-PCR?

With the one-step approach, the reverse transcriptase and DNA polymerase are premixed into a single tube. This allows the RT step and subsequent amplification step to be performed in a single reaction.

Why choose the one-step RT-PCR method?

  • Simple and fast analysis
  • Less pipetting steps to reduce errors and contamination
  • Improved data reproducibility
  • For high-throughput applications

What is two-step RT-PCR?

With the two-step approach, the reverse transcription of the RNA template is performed first. Once completed, the amplification of the cDNA is carried out in a separate reaction.

Why choose the two-step RT-PCR method?

  • Multiple targeting from the same RNA sample due to separate reactions
  • Greater flexibility to select RT enzymes and DNA polymerases for PCR separately
  • Store cDNA for later use
  • Preferred method for applications with limited amount of starting material (i.e. single cell analysis)

With so many product options on the market, it can be tough to determine which RT-PCR approach is the right one for your experiment. We hope that that these tips have helped you decide which technique to use!

For more information on these two RT-PCR methods, take a look at the video below.

Ordering information

 SuperScript IV Reverse TranscriptaseSuperScript IV First-Strand Synthesis SystemSuperScript IV VILO Master MixSuperScript IV One-Step RT-PCR System
FormatStand-alone enzyme


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cDNA synthesis kit


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cDNA synthesis master mix for two step RT-qPCR

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System for one-step RT-PCR

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