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).