Anyone working with gene expression, transcriptional regulation or cDNA cloning will undoubtedly have used reverse-transcription PCR (RT-PCR) at some point in their workflow.
RT-PCR is usually carried out as a two-step process: first, total or mRNA-enriched RNA is converted to complementary DNA (cDNA) by a reverse transcriptase (RT) enzyme. The cDNA is then used as a template for PCR.
In this post we describe an alternative set-up known as one-step RT-PCR, where reverse transcription (RT) is carried out in the same tube as the PCR reaction. Here, RNA, target-specific primers* and a one-step RT-PCR reaction mix are added to a single tube, in which RT and PCR are carried out back to back.
Because target-specific primers typically anneal to RNA at higher temperatures than oligodT or random hexamers, one-step protocols often use higher RT reaction temperatures than two-step workflows. Optimised RTs that can withstand higher reaction temperatures are also typically used in one-step setups.
*Unlike two-step RT-PCR where oligodT, random hexamers or a mix of the two are used to prime the mRNA transcripts for reverse transcription, target-specific primers alone direct cDNA synthesis and subsequent PCR in one-step RT-PCR.
Advantages of One-Step RT-PCR
As you might guess, one-step reactions are easier and less time-consuming to set up. The possibility to run RT and PCR in a single tube simplifies and speeds up the overall process, making the one-step approach attractive for high-throughout workloads with or without the aid of liquid handling systems. Other advantages include:
- High reproducibility because both enzymatic reactions (RT and PCR) take place under the same conditions, thereby reducing experimental variation.
- Fewer pipetting steps should lead to a reduction in pipetting errors.
- Closed tube format reduces contamination risk.
- Possible to run multiplex PCR on target(s) and housekeeping gene(s) from the same RNA sample.
In general, one-step RT-PCR is ideal for high-throughput screening and diagnostic laboratories where the same few genes are analysed repeatedly using well-established reaction conditions.
One-step RT-PCR doesn’t offer the same flexibility and control that is possible with two-step setups. Additional pitfalls to watch out for include:
- One-step RT-PCR may be less sensitive because it is impossible to optimise RT and PCR reactions separately in the same tube.
- Primer dimers. Since primers will be present during the RT reaction (where temperatures will be low) primer dimer formation is likely.
- Because both reactions are run back to back in the same tube, it may be difficult to troubleshoot potential RT issues.
- You won’t be left with a cDNA stock that represents your original samples, since all of the cDNA will be used during the PCR reaction. In this way, you will have to go back to the start (i.e. and redo RT) if your one-step reaction doesn’t work out.
For Successful One-Step RT-PCR
High quality RNA is critical to the success of one-step RT-PCR, since the cDNA is used for the subsequent PCR without any possibility to perform intermediate quality control steps.
The reaction conditions needed to support RT and PCR may not be optimal for either alone, and this may affect overall efficiency and yield. You may be able to get around low PCR yields by starting out with a large quantity of RNA, but bear in mind that this may increase the variation between the different RT reactions and complicate assay interpretation significantly.
One-Step RT-PCR Kits from Nordic BioSite
Nordic BioSite represents a few different suppliers of one-step RT-PCR reagents. All of the one-step kits in our portfolio have optimised buffer systems that guarantee sensitivity and efficiency for both the RT and the PCR reactions. You can find our full one-step RT-PCR portfolio here.
For anyone working with very large sample numbers and high-throughput workflows, it may be worthwhile checking out the SensiFASTTM Probe One-Step Kit from Bioline®. This kit was recently tested for COVID-19 detection and found to be so robust that even crude RNA lysates were suitable as input material instead of purified RNA (1). Have a look here for an overview and feel free to contact us if you wish to know more.
- S. Fomsgaard, M. W. Rosenstierne, An alternative workflow for molecular detection of SARS-CoV-2 – escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020. Euro Surveill 25, (2020).