Great and straightforward protocol!Do you recommend first normalizing...

CM
Cris Martinez
Nov 30, 2019
protocol Protocol: mRNA Stability Assay Using Transcription Inhibition by Actinomycin D in Mouse Pluripotent Stem Cells
Great and straightforward protocol!
Do you recommend first normalizing Ct values to an endogenous control (that should not change with Actinomycin D treatment) first in case of pipetting errors, RNA calculations etc first? If so, what would be a good control? I found that normalizing
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Minna-Liisa Änkö Author Answered Dec 4, 2019

Centre for Reproductive Health and Centre for Cancer Research, Hudson Institute for Medical Research, Australia

Glad to hear that you find the protocol useful! Actinomycin D is not a selective PolII inhibitor, so the transcription of any RNA is affected (including PolI and PolII transcripts) making it hard to choose a transcript for normalisation. Therefore, we only normalised to the first data point to determine the rate of decay. FOr this reason, it was important to run multiple independent experiments (biological replicates). There are more specific PolII/III inhibitors such as alpha-amanitin where normalization to a PolI transcript would be more appropriate. We also analysed transcripts with previously established decay rates as controls (in our case c-Myc).
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