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Old 02-02-2012, 12:20 AM   #1
Junior Member
Location: Brisbane

Join Date: Jan 2012
Posts: 7
Default Ribo-Zero before or after DNase treatment?

Hi everyone,

I have been looking at a few different protocols to process RNA samples for RNA sequencing and I am a little confused about the order of some of the steps in the process.

I have a gram-negative bacteria that I have extracted RNA from using an RNeasy midi kit. I would now like to digest the DNA and deplete the 23s/16s, however, I'm not confident with which step should be done first as some protocols I have read perform rRNA removal before DNase treatment. This could be problematic because, from what I have read about rRNA removal from even large amounts of RNA initially (~5 ug), the yield can be quite low (100 -> 300 ng). It seems it would be better to first perform the DNase digestion on the RNA and get a decent yield and then rRNA deplete the sample. Otherwise, if you do it in reverse and perform the DNase digestion last you risk losing a larger percentage of your already low yield.

Does anyone have any suggestions as to which step is best to do first?

In which order do people normally do these steps?
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