Hi,
I have isolated RNA smaller than 200 nts and I am going to sequence on the Illumina platform. I did not use DNase during the isolation step.
My question is if this is really necessary?
I have run the samples on a gel and there are no traces of genomic DNA, but of the gels are not that sensitive. Also I worry that DNase treatment might just chop up the DNA into smaller fragments and that will actually increase the contamination in my library! Further, the library prep (ScriptMiner from Epicentre) favours RNA over DNA (I think). Finally I am afraid that I will loose much RNA when I clean up after DNase treatment.
I also think that any DNA contamination is possible to identify bioinformatically (?).
Therefore I feel that using DNase is only to please reviewers and not really necessary.
Any thoughts, comments, or links to studies testing this would be greatly appreciated.
Jon
I have isolated RNA smaller than 200 nts and I am going to sequence on the Illumina platform. I did not use DNase during the isolation step.
My question is if this is really necessary?
I have run the samples on a gel and there are no traces of genomic DNA, but of the gels are not that sensitive. Also I worry that DNase treatment might just chop up the DNA into smaller fragments and that will actually increase the contamination in my library! Further, the library prep (ScriptMiner from Epicentre) favours RNA over DNA (I think). Finally I am afraid that I will loose much RNA when I clean up after DNase treatment.
I also think that any DNA contamination is possible to identify bioinformatically (?).
Therefore I feel that using DNase is only to please reviewers and not really necessary.
Any thoughts, comments, or links to studies testing this would be greatly appreciated.
Jon