Hello,
I have a couple of questions regarding small RNA sequencing for miRNAs if anyone has done it:
1. Would a regular miRNA isolation kit from Qiagen be suitable for this purpose?
2. Would the small RNA sequencing just give us miRNAs or mRNAs as well?
-In other words, if I want to look at both mRNA and miRNAs would I need to do both regular sequencing and small RNA sequencing? Or is small RNA sequencing enough to see both?
3. Are you aware of any neuroscience papers using small RNA sequencing?
Many thanks!
I did loads of small RNA seq in the context of neuroscience, so to answer your questions...
1) Yes, but it's a more expensive option and isn't really necessary. Isolation of RNA using spin columns can be adjusted via the buffer composition to change the size range of RNA that is cleaned up. Standard Qiagen RNA isolation kits will lose RNA that is smaller than about 200nt, which is why Qiagen makes miRNA kits that have different buffer formulas for when the small RNA fraction is required. The standard RNA isolation kits from most other manufacturers will capture RNA down to ~20nt - I personally like the zymo kits (they go down to ~17) but pretty much any will work, just check the size range.
2) Small RNA sequencing is ligation based and requires a 5'P and 3'OH. It will NOT detect mRNAs, or any other RNAs with a 5' cap. You will likely get some reads mapping to mRNAs for 2 reasons. Firstly, some mRNA fragments will ligate and get sequenced alongside your small RNAs. Secondly, a lot of small RNAs (including miRNAs and snoRNAs) are encoded within introns of mRNAs. The software used to annotate sequencing data is a bit iffy and one of the main types of error it makes is to annotate reads coming from these intronic small RNAs with the gene symbol of the host mRNA or lncRNA, rather than the small RNA. I had to do a LOT of manual data sanitising to get around this. To add to this, there's a general issue with small RNA databases where fragments of other RNAs have been mis-annotated as small RNAs and the error keeps getting propagated through the literature. I STRONGLY recommend that you read this paper, which does an excellent job of explaining and demonstrating the problem - it's about piRNAs but this issue is common to all classes of small RNA in my experience, although probably less so with miRNAs vs anything else, because they are better studied. https://www.nature.com/articles/s42003-017-0001-7
3) I don't usually like to cite myself on here but since it's really relevant I'll make an exception - Article Functional Interplay between Small Non-Coding RNAs and RNA M...
;. Caveats; this is a few years old now, and it focuses on small RNAs that are either modified or modifiers, but it gives some idea of what types of small RNAs are found in the brain and which have been sequenced and how. There are literally thousands of papers that have done smRNA seq in the context of neuro though, it's a well established technique in the field.If you're looking to make libraries yourself I highly recommend the NEB kits (NEBNext small RNA) which are super easy to use and in my experience produce cleaner libraries from less input than others I tried.
Hi Laura Leighton ! Legend!! Thank you so much for your answer, very well explained. Do you know of any kits that would be able to synthesise the cDNA for both small RNA seq and regular RNA seq? https://www.thermofisher.com/order/catalog/product/4475936 - I wonder if this would work?
Hi Alexandra,
Can I ask why are you so keen to detect both small RNAs and mRNAs with the same kit?
We have used small RNA kits to detect both types of molecules, you can fragment longer RNAs (e.g. mRNAs and lncRNAs) end repair and then they will be included with small RNA libraries.
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