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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.

    • What is the highest temperature the cDNA synthesis step can be performed?

    PCRAMP-021

    The highest temperature for cDNA synthesis is partly dependent on TM of the primers used. Other factors include the length of the desired amplicon and if there is potential RNA secondary structure.

    What is the recommended concentration for EpiScript RNase H- Reverse Transcriptase in RT reactions?

    221, RDXERT300

    For 1-step RT-qPCR, we have found 20-50 units per reaction to work best. In 2-step RT-qPCR, we have found 5-20 units per reaction to work best.

    What conditions do you recommend for set up of RT reactions using EpiScript RNase H- Reverse Transcriptase?

    221, RDXERT300

    For best results we recommend preparing reactions on ice for both 1-step and 2-step RT-qPCR experiments.

    Can I use random hexamer or nonamers with EpiScript RNase H- Reverse Transcriptase?

    221, RDXERT300

    Random primers can be used with EpiScript RNase H- Reverse Transcriptase. If using random primers, we suggest that the RT step is performed at 42 °C or lower as many random primers will not anneal at higher temperatures.

    Is EpiScript RNase H- Reverse Transcriptase suitable for RNA-seq library preparation?

    221, RDXERT300

    Yes, EpiScript RNase H- Reverse Transcriptase is compatible with RNA-Seq.

    Which RT enzyme, MMLV or EpiScript RNase H- Reverse Transcriptase, should I use for pathogen detection, gene expression, RNA-Seq library preparation for the human health care market and why?

    221, RDXERT300

    Both standard MMLV and EpiScript RNase H- Reverse Transcriptase can be used for these human health care applications. EpiScript RNase H- Reverse Transcriptase has the advantage of being active up to 55 °C making it more specific than standard MMLV RT enzymes.

    How should I extract or purify my RNA samples if I wish to reverse transcribe them using EpiScript RNase H- Reverse Transcriptase?

    221, RDXERT300

    MasterPure™, sbeadex™ and QuickExtract™ isolation chemistries are all suitable for use with EpiScript RNase H- Reverse Transcriptase.

    How should I extract or purify my RNA samples if I wish to reverse transcribe them using EpiScript RT PLUS?

    AS3107, MBTOOL-007, PCRAMP-104

    MasterPure™, sbeadex™ and QuickExtract™ isolation chemistries are all suitable for use with EpiScript RT PLUS.

    I left the RapiDxFire Thermostable Reverse Transcriptase (RT) out on the bench overnight. Is it still good?

    307

    Yes, the RapiDxFire RT is stable at room temperature (20 °C) for at least three months. However, we have not tested the stability of the combined reagents (buffer, template, primers) over time at room temperature.