I have some spin column left over from the kit that originally made to extract total RNA. I want to use these columns to extract microRNA. Is it possible? Has anyone tried it yet?
Dear Pedro Paulo Gattai,
You in this regars Dr. Yeşim Doğan can help
You should find the precise info (RNA sizes captured) somewhere in the documentation for the columns / the kit. The columns do not capture small RNAs and are thus not adviced for microRNA purification. As far as I am aware, everybody uses classical Trizol protocols to include the small RNA species
Dear Ayla and Johannes, thank you for your answers.
Johannes, I generally use the kit from Ambion (miRVana Paris) for extract microRNA, by the protocol of this kit I don't need use Trizol, actually if I use Trizol with the others reagents it doesn't work, I've tested it already. Instead, when use this kit I need to use the phenol-chloroform solution that came with the kit. It occurs that this kit accompanying only 20 columns, but the reagents are sufficient to do 40 extractions, so always have reagents and missing columns. Because of it I took borrowed some columns of another kit from qiagen, and I don't have certain if these columns works properly with the reagents of the miRVana kit.
I need to use the columns of the other kit (qiagen), because the enterprise that sells the miRVana kit and its columns here in Brazil doesn't have the columns at stock, so they only will delivery the columns for me in two months, and I can't wait so long for that.
Anyway, I will test extract miRNA from my samples using the column of one kit and the reagents for the other kit.
Hi, no idea for your particular case... the qiagen manuals for rneasy or qiaamp state under principle somewhere that rna molecules of 200 nt or longer are being purified., which does not sound optimal for you.
https://www.qiagen.com/ch/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6faß33e24&lang=en
I am afraid that is not possible to use this kit to extract miRNA since the columns are not prepared to capture small RNAs.
Maybe if I use the TRIzol method for extract total RNA, is it possible? I've read here in researchgate that if I use 80% ethanol to wash the samples I can extract miRNA by TRIzol method as well.
As far as I know, the QUIamp spin columns do not bond small RNAs - they will be present in flow-through, which is why I don't recommend using this columns for microRNA purification. I used mirVana kit from Ambion and it worked perfectly with such recalcitrant cells as unicellular algae. RNeasy from Quiagen didn't perform that well, especially in terms of miRNA recovery.
I've tried loads of extraction methods (I'm going to do some miRNA sequencing) and I would not recommend trizol because it always affected RNA quality. For me, the CTAB-lysis + acid-phenol:chloroform extraction worked the best and the electrophoresis showed as high amount of small RNAs as after mirVana (in equal concentrations).
Natalia, I've tried the TRIzol method for extract microRNA and the RNA quality is similar to it that I extract with miRVana method. Besides that, the qPCR efficiency were better by TRIzol method than miRVana. So I think that the TRIzol method, or conventional method, is sufficient to extract microRNAs. In other words, I don't need any column to extract in an efficiently manner microRNA from the sample. It is just extract total RNA by the TRIzol method that you will extract microRNA also.
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