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Old 06-25-2012, 04:45 AM   #6
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,315
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Listen, you can attempt to convince yourselves that DNAse treatment is not necessary all you want, but that does change the fact that failing to do it can result in DNA sequence contamination of your RNAseq results.

Also, if your RNA is degrading during DNAse treatment, the simple explanation is that your preps still have RNases in them. That is, your RNA prep has been unsuccessful at completely removing them. So, even if you skip the DNAse step, to spare your RNA, all you accomplish is a circumvention of the QC step -- your RNA will likely degrade during a later step.

A "standard paradigm protocol" would have you isolate intact RNA free of contaminating RNases and depleted of most DNA. Then DNase treat to remove the remaining DNA, followed by some purification to remove the DNase and oligonucleotides. (I like Zymo columns for this purpose.)

No one likes to see their RNA degrade during DNase treatment, but better it happens there than at a later step where library QC may not catch it.

--
Phillip
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