Question: Differential expression in qPCR Data after filtering
0
gravatar for peter pfand
3.3 years ago by
peter pfand80
Germany
peter pfand80 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 • 1.1k views
ADD COMMENTlink modified 3.3 years ago by Devon Ryan91k • written 3.3 years ago by peter pfand80
1
gravatar for Devon Ryan
3.3 years ago by
Devon Ryan91k
Freiburg, Germany
Devon Ryan91k 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 COMMENTlink written 3.3 years ago by Devon Ryan91k

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 REPLYlink written 3.3 years ago by Anima Mundi2.4k

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 REPLYlink written 3.3 years ago by Devon Ryan91k
0
gravatar for Anima Mundi
3.3 years ago by
Anima Mundi2.4k
Italy
Anima Mundi2.4k wrote:

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

ADD COMMENTlink written 3.3 years ago by Anima Mundi2.4k
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