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Hi,
We're going to do a RNA-SEQ experiment (with Nextseq 500) with bacterial RNA for differential gene expression. We have cells lysed and frozen in TRIZOL, and now we're using ZYMO's Direct-Zol kit to extract and purify RNA. DNAse I was used in column (20 min. at ~28 oC). After elution, samples were tested in QUBIT using dsDNA broad range reagent, and we got 2-3 micrograms/mL of DNA. RNA concentrations read in Nanodrop are about 80 micrograms/mL.
The question is, should we worry about traces of DNA in the samples? How much DNA contamination is "allowed" in RNA samples for RNA-SEQ, without interfering with results?
If there's anyone using similar reagents, we'd be glad to hear about modifications of the protocol, say, longer DNAse I treatment etc.
By the way, we had other samples extracted and purified with the same protocol that we tested for DNA contamination using PCR (before we got QUBIT), and we detected no amplicons. We are yet to test the current samples with PCR.
Thanks.
We're going to do a RNA-SEQ experiment (with Nextseq 500) with bacterial RNA for differential gene expression. We have cells lysed and frozen in TRIZOL, and now we're using ZYMO's Direct-Zol kit to extract and purify RNA. DNAse I was used in column (20 min. at ~28 oC). After elution, samples were tested in QUBIT using dsDNA broad range reagent, and we got 2-3 micrograms/mL of DNA. RNA concentrations read in Nanodrop are about 80 micrograms/mL.
The question is, should we worry about traces of DNA in the samples? How much DNA contamination is "allowed" in RNA samples for RNA-SEQ, without interfering with results?
If there's anyone using similar reagents, we'd be glad to hear about modifications of the protocol, say, longer DNAse I treatment etc.
By the way, we had other samples extracted and purified with the same protocol that we tested for DNA contamination using PCR (before we got QUBIT), and we detected no amplicons. We are yet to test the current samples with PCR.
Thanks.
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