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Asked 5th Sep, 2020

What is the most probable theory about the Sumerian origin according to you and on what reason your answer is based?

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How to solve these problem involving lowly expressed genes in qPCR?
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6 answers
  • Kent FungKent Fung
Hi, so I have a few questions on the normal practice people use for qPCR analysis inovlving gene with low expression
1) How do you replace undetermined value? I have seen people using a MLE (which is a bit too complicated for qPCR analysis in my opinion ( ). Another way is just to replace it with maximum Ct value (usually 40) which will induce bias for obvious reasons. Is there a third method you would suggest especially for t-test or other statistical analysis?
2) Some low expression genes have a Ct value between 35 to 40 in one or two of the 3 technical replicates on the same plate. Would you eliminate the value and just replace it with 40 or whatever you used for your undetermined Ct value? This is especially important in doing statistical analysis if you have 35 then two undetermined wells.
3) For samples with 3 very high Ct in their technical triplicates due to low expression (this is pretty common from my experience and would lead to high SD if any analysis is to be performed) what would you do? Would you set a threshold at like 35 and just say everything above 35 is undetected or are there other ways to deal with it?
I know some of you might suggest repeating the experiment but sometimes it just won't conform when the expression is really low and especially when the sample is clinical sample. Unlike cell line I find them more variable especially with genes with low expression.
What are your opinions/suggestions?

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