View Single Post
Old 02-02-2012, 12:20 AM   #1
colin.aibn
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?
colin.aibn is offline   Reply With Quote