I have performed qPCR on a tissue, where I have extracted total RNA and also purified mRNA. I ran the qPCR samples together, and have therefore been exposed to the same conditions except the purification step from total RNA -> mRNA. In my results, a gene is down-regulated in the total RNA, but it is not in the mRNA. What could be the reason for that?
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$\begingroup$ What is the mean magnitude of cycle number difference between the treatment and control in each case? I would favor the total RNA results since mRNA enrichment has different biases for different transcripts. $\endgroup$– acvillDec 18, 2021 at 14:38
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$\begingroup$ @acvill the mean magnitude difference of the RQ values for the total RNA is 6.63, and for the mRNA -0.8957 - I have subtracted the treatment from the Control $\endgroup$– F.SalDec 18, 2021 at 14:58
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There are a few possibilities.
- For whatever reason, your transcript of interest is poorly captured by your mRNA enrichment such that, in both the control and the treatment, mRNA enrichment selects against your mRNA. This would be evident if the CT value for the mRNA control is much higher than the total RNA control, assuming that the mass of total RNA you used in your mRNA enrichment is the same as the mass of total RNA used in each qPCR replicate.
- If your primers / probes are nonspecific, your total RNA qPCR results could be the result of downregulation of some off-target noncoding RNA. If your mRNA enrichment protocol is non-destructive (i.e. relies on positive selection of mRNA and not degradation of non-mRNA), you could test the specificity of your primers / probes by performing qPCR on your non-mRNA fraction. Though, interpretation of these results could be confounded if point #1 is also true.
