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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.

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.

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.

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.

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.

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.