I have a fraction on my RNA blot less than100nt which I guess to be microRNA. I have no idea which kits are suitable for small RNA extraction. I got this RNA along with my main viral RNA using viral RNA extraction kit from QIAGEN. Would it be enough to use this kit or should I use a special one for small fragments? Also to be sure about the identity of those fragments I want to sequence them. Apparently, they should be cloned in ssDNA followed by making cDNA. So, which kits are working best for it?
I have no experience in sequencing; but I recently tried mirVana™ miRNA Isolation Kit, from LIFE TECHNOLOGIES, and I got miRNA with very good integrity.
Good luck!
mirVana™ miRNA is a good kit. However if you prefer doing that without using phenol, I would recommend miRNA purification from Norgen Biotek Corp.
http://norgenbiotek.com/display-product.php?ID=65
I hope this is helpful.
We feel no need for miRNA purification. We always use total RNA (including mRNA, lncRNA, miRNA, etc.) for all our gene expression work, either microRNAs or longer RNAs.
Dear Jo, what method have you found useful? is it a column-based or phenol-based like Trizol? because some manufacturers say that you are likely to lose small RNAs by using ordinary column-based kits. Meanwhile, I know that Trizol is good for this purpose except that its phenol content, if remained, may cause a problem in qPCR reactions.
We are very successful with Qiagen's miRNeasy micro kits. It's a combination of Qiazol (Trizol-like) and column with special conditions to retain small RNAs. More information on our workflow on http://www.biogazelle.com/lab-services/applications/mirna
Thanks for the answer Jo, but I assume this kit is specifically made for miRNA isolation like the Roche's miRNA isolation kit which I usually use and is very good. I just thought you meant kits like Qiagen's RNeasy or similar products. Anyway, personally I recommend Roche's High Pure miRNA isolation kit which has two protocols, one for isolating only small RNAs and one for total RNA including small RNAs. It is only column-based and very efficient. I used it for both cell cultures and frozen tissue successfully.
No, miRNeasy is for total RNA isolation, small and long transcripts.
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