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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.

What is the longest cDNA product that RapiDxFire Thermostable RT can synthesise?

307

RapiDxFire Thermostable RT has successfully synthesised products up to 1kb in 5 minutes.

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.

    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.

    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.

    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.

    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.