I have isolated RNA shorter than 200 nts. On an agarose gel I cannot see any genomic DNA, but this is not very sensitive. I do not want to treat with DNase because I am afraid of three things:
1) I may chop up the long DNA into smaller bits that will more easily be included in my sequence library.
2) The DNase may also degrade my RNA.
3) I will loose a lot of RNA when I clean up after the DNase treatment.
The library prep method I am using (Scriptminer) I think will favour RNA over DNA in the adaptor ligation and low temperatures are used throughout. Does anyone have any comments or suggestions? Or any references to studies testing the amount of DNA contamination in small RNA libraries?
I would still treat the sample with DNase I. Do not be afraid of losses. If you use an RNase inhibitor to protect your RNA, and glycogen to coprecipitate it, there will be no harm. Test again the quality of your RNA after treatment and ship. (Just in case, I routinely use DNase I from Thermo Scientific with the supplied buffer, 37°C for 30 min, then PCI extraction.)
Jon Bråte Did you end up testing this? I have the same question particularly because I am working with biofluids which do not contain a lot of small RNA anyway!
Hi Catarina Castanheira , this is so long ago I don't remember exactly what we ended up doing. Now we usually don't make cDNA libraries ourselves and we always isolate totalRNA, treat with DNase (usually not on-column treatment, but after the isolation and elution) and then just ship the sample to the sequencing facility.
I am hesitant to advice you not to treat with DNase, but I don't really see how DNA can end up in your sequencing library. So personally I wouldn't be too afraid. But perhaps you can test on a few samples and see how much RNA you loose?
Hi Jon Bråte ,
you do the DNAse treatment after elution and ship the samples for library prep, without another clean-up step? Do you also use a stop solution?
I am just wondering, as we would like to do the same but we also use a stop solution (for inactivation of DNAse) and I was thinking that might make problems in library prep?
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