You do not have to, but it helps and also depends what you mean “small RNA”. When you build small RNA NGS library size selection is very important, otherwise you reads will represent all transcriptome, and small RNA will be extremely small part of data.  You can use for example LifeTechnologies mirVana kit to enrich you sample in small RNA. You just treat total RNA with this kit, but it is not perfect solution, this kit enrich for RNA smaller then 200 nt, so for micro RNA library there will be still a lot of reads larger then desired. This kind of problem recently has friend from our group and he decide to go for size selection on polyacrylamide gel prior library building. Anyway he did not want to build library himself, he was looking for NGS company in Europe, it was not easy and finally he choose BGI service. Best regards, MK.

Hi Javier,

the first thing I would like to know is the downstream application and what kind of RNAs you are looking for. Depend on you answers I would recommend doing the size fractionation by gel and would addionally trying to deplete the rRNAs by using a commercial kit.

If you want to do NGS than maybe it is better to look for a company doing the size fractionation, rRNA depletion and  library preparation as they are more experienced with this. For NGS it is necessary  to have consistent library preparation.

All the best,

Ulrike

Hi Ulrike,

I am currently doing libraries from microRNAs for NGS and I'm working with Truseq Small RNA kit Illumina in this kit tells me to purify the libraries must run a polyacrylamide gel and cut the band. This procedure is wasteful so I would like to use magnetic beads for purification.

do you do an inhouse sequencing or what is the reason for doing it youself? The size fractionation via PAA gel is pretty efficient.

Hi,

magnetic beads clean-up is alternative approach to gel-selection, which use LifeTech in their Ion Torrent RNAseq kits. As an input you may use total RNA, or better is to do 2 step size selection: 1/ small RNA enrichment by mirVana (up to 200 nt) then 2/ magnetic beads size selection of RNA in range 10-40 nt. It is not a problem to “mix” approaches, kits etc. If for example Illumina prefers PAGE selected RNA, it doesn’t mean that you cannot use for Illumina as an input RNA selected in LifeTech’s way, if you are able to assure amounts and quality of RNA that needs Illumina library kit. I think that in your case rRNA depletion is pointless. OK, for whole transcriptome is crucial, but is extremely expensive and just removes rRNA, leaving other long ncRNAs or mRNAs, so in fact do not concentrate small RNA. Magnetic beads module that is used in LifeTech kits you can buy separately, I was using this and works quite OK. It is based on differences in elution efficiency of small and larger RNA depending on ethanol concentration. So it is important to have absolute – 100% pure alcohol and do very careful pipetting of it, with few steps of tip pre-wetting (everything is in manuals). As 100% alcohol is very hygroscopic I prefer to aliquot it, store parafilmed tubes in a freezer and use them once. There you have links to protocols.

Best regards, MK.

https://tools.lifetechnologies.com/content/sfs/manuals/4476286.pdf

https://tools.lifetechnologies.com/content/sfs/manuals/4476289.pdf

Thank you Michal

Someone knows or has worked with this kit miRCURY ™ RNA Isolation Kit-Tissue, as is its performance in microRNAs recovered?