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Why is my reverse transcription reaction not creating cDNA?

307

  • Primer issues
    • The gene-specific primer design is incompatible with high reaction temperatures
    • The gene-specific primer is not complementary to the target of interest.
    • Non-specific primers were used with a Tm that is too low
  • The incorrect reaction temperature was used.
  • Contaminants in the sample preparation interfered with the reaction conditions.
  • The reaction buffer conditions are not correct. Use the kit-provided 10X Thermostable RT Buffer provided with the kit.

    • Can I shorten the activation time for the heat-activated thermal stable reverse transcriptase?

    PCRAMP-021

    Shortening the activation time for the heat-activated reverse transcriptase is not recommended. We recommend a three-minute activation time to fully activate the reverse transcriptase. However, assay optimisation may enable shortening the activation time to a minimum of one minute depending on other factors such as other thermocycling parameters, sample type, and sensitivity requirements.

    Can I store the extracted RNA before performing cDNA synthesis?

    QER090150

    Yes. The extracted RNA can be stored at –80oC for up to 1 year. Divide the extracted RNA into small aliquots before storage to minimise the amount of freeze-thaw cycles.

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