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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.

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

30260-1
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 Lyo-Ready EpiScript Thermostable RT?

30260-1
Column-based and magnetic bead-based isolation chemistries are both suitable for use with Lyo-Ready EpiScript™ Thermostable Reverse Transcriptase.

What is the optimum temperature for Lyo-Ready EpiScript™ Thermostable Reverse Transcriptase?

30260-1

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?

30260-1
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.

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

30260-1
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.

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.

    • Which RT enzyme (MMLV, EpiScript RNase H- Reverse Transcriptase, or EpiScript RT PLUS) should I use for pathogen detection, gene expression, or RNA-Seq library preparation, and why?

    AS3107, MBTOOL-007, PCRAMP-104

    All three reverse transcriptases; standard MMLV, EpiScript RNase H- Reverse Transcriptase and EpiScript RT PLUS can be used for these 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. EpiScript RT PLUS provides the added benefit of allowing room temperature reaction setup.

    What is the difference between EpiScript™ RNase H- Reverse Transcriptase and EpiScriptT RT PLUS?

    AS3107, MBTOOL-007, PCRAMP-104

    EpiScript RT PLUS contains a blocking mechanism that suppresses reverse transcriptase activity at lower temperatures allowing for room temperature reaction setup.