Estimating activity units and quality controls on reverse transcriptases

Nucleus
1

Hi All

How are people figuring out activity units and quality controls for RTs? It would be great to have some standard assays we can run across different batches and labs.
This apparently came up in discussions on the last Reclone call!

Jenny

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Hi Jenny, I think this can be a good starting point, EnzChek™ Reverse Transcriptase Assay Kit.

It’s essentially using PicoGreen as probe (binds dsDNA), in a reaction started from oligo dT and a polyA template. The key is performing a standard curve using e.g. lambda DNA in this case.

I guess it should be easy to work towards an open kit from here: oligo dT is available mostly in all labs, have to find/provide a template containing a polyA that would produce sufficiently long transcripts, and make sure a quantified standard is available also to create the standard curve. But it will require labs to purchase PicoGreen (and have a fluorescence reader).

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