We are trying to purify RNA from tissues, which are preserved in a proprietary solution (don't want to disclose the name of the commercial kit for fear of a free advertising).
One key feature of the kits is that samples can be preserved at r.t. for a long time - weeks - (and thus can be shipped to collaborators without worries).
According to their protocol, the samples need to heat up at 50C for longer than 2 hrs (which seems to be breaking the cells and releasing nucleic acids). Following 50C incubation, samples are incubated at 90C for 15 min and then cooled to r.t. A proprietary neutralizer solution is then added and samples are put on ice for 10 min. Samples are then centrifuged and sup is collected for ethanol /isopropanol precipitation.
My question is regarding the 90C incubation and the neutralizer solution. I went through many other traditional protocols for RNA purification and none requires 90C incubation (some even warn not to put samples in high temp). Any idea what this step is for? Based on the principles of RNA purification, is it feasible to mix samples in the "preservation solution" with phenol-chloroform or with Trizol (??) and proceed with the conventional protocols to purify RNA?
Any suggestion/comment to share will be greatly appreciated. Thank you.
Have you tried asking the manufacturer first? Since this is an unnamed, proprietary solution, one can only speculate whether what you are proposing will work.
Sounds to me like it is either a de-activation step, or a partial lysis step to weaken the membranes for the 'neutralizer' solution.
As Andrew suggests above, contact the manufacturer to get some input. In my experience with TRIzol, adding heat is a bad idea and leads to RNA degradation. Without knowing what the proprietary solution is, there's no way to know whether mixing it with TRIzol will ruin your samples and your day or work just fine.
Additionally, if you're keeping the tissue in RNAlater or its equivalent, I've never needed to add a heat step, whether extracting RNA by TRIzol or a kit.
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