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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.

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.

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.

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.

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 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.