Hi, I work with viral RNA extraction from fecal samples. Initially, it is necessary to prepare a 10% fecal suspension in 1X PBS or 0.01M Tris Hydrochloride Ca++ pH 7.2 solution, centrifuge for 10 minutes at 4000 rpm. After, take out the supernatant and extract the viral RNA from it using any commercial kit for RNA isolation, following the manufacter's recommendation.

I hope i helped you.

Thanks for your answer, Dielle. I don't know exactly what is in the stool matrix, but here is what the manufacturer says "Supplied in stool diluent/matrix that mimics composition of true clinical specimen."

I centrifuged after the pro K and Buffer RLT incubation so I'll try getting a supernatant before anything else on my next run. My concern is that I'm using the RNeasy Mini kit for this when there is no "from fecal" protocol and they have their own PowerFecal kit that has very different reagents in the kit. In your experience, do you make additions or changes to non-specific RNA extraction kits that optimize your results? Or maybe the pro K digestion is unnecessary?

I usually prepare a fecal suspension before extraction and then I follow with the extraction kit from the lysis step. Most commercial RNA isolation kits do not use proteinase K, so if your goal is to obtain RNA, this step may not be necessary. Go straight to the lysis step, with the recommended buffer in the kit, and follow the column binding - wash - elution steps.

If you're starting with a raw stool sample, maybe also that's the problem.