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5 months ago
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I was wondering the state of the art and the opinion and experience of the community in terms of the best kits for RNA extraction from different fish tissue (for annotation and gene expression analysis eventually).
Not a fish person here (and Q is actually off-topic) but I think RNA extraction itself will work with any standard RNA kit. What is more important is that you make sure to have a library prep kit suited for fish. Most kits are geared towards human and mouse RNA when it comes to fragmentation of full-length RNA. Be sure to contact sequencing facilities and ask whether they have experience with that sort of RNA. They might even give recommendation for extraction protocols. Or see what is in the method sections of papers.
Yes, a standard extraction (e.g. homogenize + chloroform-phenol) will work. Not sure why a library prep kit would need to be specifically suited for fish (it shouldn't be).
In the kits we use for mouse/human the fragmentation step (magnesium+heat) has incubation times optimized to result in about 200nt-ish fragments, and that depends on the average total length of mRNAs. Is that the same for fish? I have no idea.
Not a special kit per se, but incubation times and recommended amount of PCR cycles given x ng of input RNA is probably (?) not the same for any given species.
I see; so the conditions that need to be optimized. Makes sense.
Conditions should be similar for fish (at least if we're talking about zebrafish here), but library quality on an Agilent machine will tell you anyway.