Question: Uneven PCR in RNA-seq Preparation
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gravatar for biostar.anon
4 weeks ago by
biostar.anon0 wrote:

Is it normal practice to prepare mRNA-seq libraries with uneven PCR to compensate for low RNA input ? and if so how is it treated in analysis and communicated in article ?

rna-seq • 118 views
ADD COMMENTlink modified 4 weeks ago • written 4 weeks ago by biostar.anon0
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Can you clarify where this question is originating from? Generally kit manufacturers will have very specific instructions on input requirements/number of PCR cycles one can use with low RNA inputs (an example here).

ADD REPLYlink written 4 weeks ago by genomax65k

Yes while it is true that there's a protocole, I am currently in contact with a sequencing facility which states their standard method for if a DNA converted mRNA has too little molarity/ [] in QC is to amplify 5 supplementary cycle. Has anyone heard any similar protocol ?

ADD REPLYlink written 4 weeks ago by biostar.anon0
1

As long as they are treating all of your samples identically it may be ok. You will need to be mindful of PCR duplicates though they would be hard to completely eliminate if you don't have UMI's.

Please use ADD REPLY/ADD COMMENT when responding to existing posts to keep threads logically organized. SUBMIT ANSWER is for new answers to original question.

ADD REPLYlink modified 4 weeks ago • written 4 weeks ago by genomax65k
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