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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.

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.

How should I extract or purify my RNA samples if I wish to reverse transcribe them using EpiScript RT PLUS?

AS3107, MBTOOL-007, PCRAMP-104

MasterPure™, sbeadex™ and QuickExtract™ isolation chemistries are all suitable for use with EpiScript RT PLUS.

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.

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.