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Normalization issues

Posted by cdarwin102 on 11 Feb 2020 at 11:19 GMT

Very nice study. However, there is an issue, hopefully the authors can comment.

TRAP method pulls down a lot of non-specific mRNAs, presumably non-specifically binding to beads, Ab or the tube. So, one has to first normalise say the cre+ive TRAP sample to the cre- control to remove this background. Then, one can calculate TE by using the transcriptome as the denominator. Without this cre-ive control normalization, one isn't looking at the true translatome.

In the ribosome profiling method, you are kind of ameliorating for this background by digesting with RNAse which is presumably degrading the background mRNAs thus giving a more accurate picture of ribosome bound mRNA. In essence, your ribosome profiling method is TRAP plus RNAse digestion, which is different from traditional sucrose gradient dependent ribosome profiling- a far more crude method, that gets ribosomes from heterogeneous cells.

No competing interests declared.