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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.

Can I use EpiScript RNase H- Reverse Transcriptase for monitoring gene expression in mammalian cells?

221, RDXERT300

EpiScript RNase H- Reverse Transcriptase is very useful in the monitoring of gene expression and can be used with both probe hydrolysis and gene intercalation methods of detection.

What is ‘template-switching’, and does EpiScript RNase H- Reverse Transcriptase have this capability?

221, RDXERT300

Template-switching is the addition of non-template nucleotides to the 3′ end of the transcript, followed by annealing of a primer. This is followed by second strand synthesis by the reverse transcriptase using its innate DNA polymerase activity. EpiScript RNase H- Reverse Transcriptase does have this capability.

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.

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.

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