I have a very basic question as mentioned in the question when RNAseq technology measures the mRNA abundance if it is acquired after degradation of dsRNA by miRNA or before? I would like to clarify if the primary change (contributed by miRNA) is observable at the mRNA level or protein level? or detailed answers to different views/possible different mechanisms involved are welcomed.
This question is wrt Illumina tech.
miRNAs usually don't work through mRNA degradation but rather by blocking translation. So for majority of cases you would see the action of miRNA at the protein level (but most papers in transciptomics tract miRNA effects by measuring the levels of mRNA, which is a bit weird). However the lowered amount of protein can be used as signal which affects the amount of its mRNA expression.
Also thinking about pre or post miRNA action is a bit misleading. You cannot turn on some kind of clock that at time 0 equals the situation where no miRNA is present. Pretty much you can tract what happens in response to some action: you change the level of mRNA and look at what happens to its putative miRNA regulator.
Hi there
Interesting question. You have to take into account that when you perform a whole cell NGS transcriptomic analysis you are dealing with an average of events that are happening in all your cell population. So, in some cells you can have different levels of a particular mRNA transcript, that are averaged when you consider the whole population.
Saying that, miRNAs main action in animals is exerted by a translational blocking and consequently observed as a decrease in the corresponding protein levels. It is also a fact, that when a miRNA is acting over a mRNA transcript for a long time, the cell will degrade it, so you will also see a decrease in mRNA levels for those transcripts that are actively regulated by miRNAs. This is the reason because many authors used the mRNA levels to infer miRNA-mRNA regulatory relationships. However the most correct way to address that would be to use proteomic data, which is not so easily available, more expensive to get than transcriptomics, and sometimes not so easy to interpret. So, in conclusion, the reasons for using mRNA levels to check for miRNA regulation are both technical and physiological.
I routinely use an approach that considers the UP-regulated miRNAs and the negatively correlated DOWN-regulated mRNA targets to built up regulatory networks. The opposite (DOWN-regulated miRNAs and UP-regulated targets) is not so easily to interpret, since the UP-regulation of a mRNA transcript would be due to more strong reasons than the absence of its regulatory miRNA (namely presence of transcription factors).
Hope it helps. Happy to continue the discusion
All the best
Paco
Ireneusz Stolarek and Francisco J. Enguita Thanks for the explanation. If I have low protein expression in one condition compared to other but mRNA expression is the same. At the same time, I have data of miRNA expression measured by small RNASeq from the same experiment. So I am trying to justify this through miRNA expression. That's why I have this question in my mind. It seems this explanation could be possible.
Amarinder Singh Thind, .... it sounds plausible. Check if you have some putative targeting miRNAs to your mRNA transcript, and maybe you can build some story about.
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