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What is the difference between EpiScript™ RNase H- Reverse Transcriptase and Lyo-Ready EpiScript™ Thermostable Reverse Transcriptase?

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Lyo-Ready EpiScript™ Thermostable Reverse Transcriptase (Lyo-Ready EpiScript Thermostable RT) is an MMLV variant engineered to have increased thermostability over Episcript. It is also formulated to be compatible with lyophilization.

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

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

Why is my reverse transcription reaction not creating cDNA?

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  • 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 optimum temperature for Lyo-Ready EpiScript™ Thermostable Reverse Transcriptase?

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    The optimum temperature for Lyo-Ready EpiScript™ Thermostable Reverse Transcriptase is dependent on a range of factors. These include the target sequence and structure, the type of reverse transcriptase (RT) primer that you are using, and whether you are doing 1-step or 2-step RT-qPCR.

    If your target sequence has a high %GC content or a high secondary structure, higher temperatures of 50 °C to 60 °C are better. If random primers are being used in the RT reaction, we do not recommend using temperatures above 42 °C as many hexamers and nonamers will not anneal at 50 °C or 55 °C.

    What is the recommended concentration for Lyo-Ready EpiScript™ Thermostable Reverse Transcriptase?

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    For 1-step RT-qPCR, we have found 100 units per reaction to work best but an enzyme titration between may be needed to optimize the enzyme concentration that is best for your primers and template.

    What is the optimum temperature for EpiScript™ RNase H- Reverse Transcriptase?

    221, RDXERT300

    The optimum temperature for EpiScript RNase H- Reverse Transcriptase is dependent on a range of factors. These include the target sequence and structure, the type of reverse transcriptase (RT) primer that you are using, and whether you are doing 1-step or 2-step RT-qPCR.

    If your target sequence has a high %GC content or a high secondary structure, higher temperatures of 50 °C or 55 °C are better. If random primers are being used in the RT reaction, we do not recommend using temperatures above 42 °C as many hexamers and nonamers will not anneal at 50 °C or 55 °C.

    In standard 1-step RT-qPCR experiments (non-challenging targets), we found minimal differences in performance when using either 42 °C, 50 °C and 55 °C for incubation. In 2-step RT-qPCR, however, we noticed that higher temperatures for the RT reaction resulted in reduced Cq values.

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

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

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

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    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.

    Can I use random hexamer or nonamers with Lyo-Ready EpiScript™ Thermostable Reverse Transcriptase?

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    Random primers can be used with Lyo-Ready EpiScript™ Thermostable 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.

    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.