DNase treat RNA before SOLiD Small RNA?

[DrDTonge]

Dear all,

As the title suggests I am running a batch of small RNA sequencing using the SOLiD system. Having extracted good quality total RNA (RIN >9), the yield of some samples is rather low (as low as 20ng/ul). Therefore I'm trying to find out whether gDNA could possibly affect small RNA sequencing in an attempt to avoid this step and the likely sample loss.

My thinking is that I perform the ligation reaction (which favours single stranded species) and then following cDNA synthesis, size select to only include the miRNA size range. Given that gDNA is huge, and that I don't perform any sort of digest or sonication, I am right in thinking there is a limited chance of me including any gDNA in my library.

Happy to be completely wrong...!

D :-)

[DrDTonge]

Going to give the DNase treatment a shot with a single sample to determine the RNA recovery post precipitation. I think I'd be happier running DNase prior to library prep so here goes...

[pmiguel]

Probably you are right. Actually if your DNAse treatment does not go to completion you might increase the amount of DNA in the size fraction you are attempting to isolate.

--
Phillip

[DrDTonge]

Thanks very much for your input.

I have run a trial DNase treatment and am unhappy with the recovery of small RNAs following precipitation.

Your comments with regards incomplete DNase digestion are very interesting. In fact this was my greatest concern as incomplete gDNA digest could leave DNA species at the correct size for inclusion following cDNA size selection.

Best wishes and thanks once again,

D