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Question: Differential expression in qPCR Data after filtering
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gravatar for peter pfand
2.8 years ago by
peter pfand • 80
Germany
peter pfand • 80 wrote:

Dear community,

I have qPCR data samples with a lot of noise and a general low amplification. Many Ct values are set therefore to 40 (or NA, I'm using R package HTqPCR). My question is whether I should filter out "genes where the results are of low quality" before performing DE analysis, and how I can trust the p-values. Are results not going to be misleading if I significant fraction of the genes is removed from the analysis?

Thanks in advance
differential expression qpcr • 1.0k views
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modified 2.8 years ago by Devon Ryan ♦ 88k • written 2.8 years ago by peter pfand • 80
1
gravatar for Devon Ryan
2.8 years ago by
Devon Ryan ♦ 88k
Freiburg, Germany
Devon Ryan ♦ 88k wrote:

You can go ahead and discard the Ct ~40 genes, you don't have the ability to measure any changes in them to begin with. The results won't be misleading if you remove them.
ADD COMMENT • link written 2.8 years ago by Devon Ryan ♦ 88k

Hi Devon, I see your point. However, what if a gene becomes measurable (or unmeasurable) only after the treatment? Also, if he ends up removing, say, 60% of the studied genes, the overall conclusions might be misleading (e.g. he might think his treatment favors up-regulation of a certain class of transcription factors, that were instead simply comparatively more easy to measure).

ADD REPLY • link written 2.8 years ago by Anima Mundi • 2.4k

If only one group has such a high Ct then that's not a problem. Filter by the average or median Ct across groups.

ADD REPLY • link written 2.8 years ago by Devon Ryan ♦ 88k
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gravatar for Anima Mundi
2.8 years ago by
Anima Mundi • 2.4k
Italy
Anima Mundi • 2.4k wrote:

Yes, they are going to be misleading. I would rather try to optimize your wetlab workflow (e.g. changing PCR primers).

ADD COMMENT • link written 2.8 years ago by Anima Mundi • 2.4k
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