I use the RNeasy kits from Qiagen for my RNA extractions. I follow the protocol, however normally perform my gDNA elimination step once I have eluted my RNA. I then DNAse digest using a kit from Invitrogen (Life Technologies) as per manufacturers protocol. I then reverse transcribe my RNA for qPCR.
I wondered how many of us perform the on-column digestion step as recommended by Qiagen? If so, how do you rate your RNA quality (I only have access to a Nanodrop & sadly not a Bioanalyzer) and your yield? Do you prefer on-column or post-elution, do you think it makes any difference? Does anyone mix and match other manufacturers, eg RNeasy on-column digestion with Invitrogen DNase?
Has anyone tried the new RNeasy kit which has a special gDNA eliminator column (I'm slightly dubious about this as I'm working on a rare cell population and wonder how much RNA I'll loose running things though another column...)?
Does anyone perform a purification/clean-up step following DNAse digestion?
Any suggestions/questions/comments gratefully received
Edited by Bunsen Honeydew, 28 March 2011 - 09:20 AM.