When do I choose a 1-step kit and when do I choose a 2-step kit?

1 step kit involves including the reverse transcriptase step in the same tube as the PCR reaction and the 2-step kit involves creating cDNA first.

1 step:
Using gene specific primers, one-step real-time RT-PCR such as the One-Step kits offer a quick and simple method to detect mRNA and so are useful when analyzing a few genes over a large number of samples as less pipetting and sample manipulation reduces variation and potential contamination. However reaction conditions needed to support both the RT and PCR may not be optimal for either reaction and it is not possible to archive the cDNA produced during the reverse transcription reaction.

This method is quick to set up and makes processing multiple RNA samples easy (especially when using liquid handling robotics), when you are amplifying only a few genes of interest. It is therefore ideal for high throughput screening laboratories where only a few assays are run repeatedly, using well-established reaction conditions, with the added advantage that multiplex PCR of the gene of interest and control genes can be done in single well.

2 step:
Two-step real-time RT-PCR, offers a truly accurate determination of mRNA and is useful when analyzing a large number of transcripts over a few samples. SensiFAST™ kits have flexibility in the priming strategy, allowing for oligo-dT, random primers or gene specific primers and are generally more sensitive than one-step as the RT and PCR occur separately and can be optimized individually. Also, the cDNA produced is more stable than the initial RNA sample and can be more easily archived for future use.

With two-step real-time PCR, the use of several tubes means that it is more time consuming and less adaptable to liquid handling robotics and so more difficult to adopt for high throughput screening assays. The use of several tubes and pipetting steps also exposes the reaction to a greater risk of DNA contamination

  • Advantages
    – Accurate representation of target copy number
    – Simple and rapid Fewer pipetting steps (reducing possible errors and  contamination)
    – Best option for high-throughput screening
    – Best method when only a few assays are run repeatedly
    – Multiplex PCR of gene of interest and control can be done in single well, from same RNA sample

  • Disadvantages
    – Usually less sensitive as it is impossible to optimize the two reactions separately
    – Difficult to troubleshoot RT step
    – No stock of cDNA

Before choosing you should consider the best one for your application, these include the ease of use and cost of reaction to the resulting yield and sequence representation.

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