Tuesday, May 27, 2014

RNA extraction - insoluble gel-like pellet

The Llama and I were working on some RNA extractions recently from a series of developmental stages in sorghum. Our lab's typical RNA extraction protocol (a GITC-phenol-chloroform based protocol; TRIzol and friends) worked fine for the developmentally younger tissue samples, but all of the more mature tissue samples ended up with a fairly large, insoluble, gel-like pellet at the end of the extraction. We initially believed the pellet to be RNA that we hadn't properly de-salted or that we had over-dried, leading to decreased solubility. However, additional de-salting, heating, physical beating, etc. never managed to resolubilize the pellet. After quite a bit of troubleshooting we determined that the insoluble pellet was starch (or at least some kind of carbohydrate) that, due to its high abundance in the tissues, wasn't removed during the extraction (of note, I had also occasionally run into a similar problem in Neurospora).

Once we had an idea that it might be a starch problem, the Llama found some specific extraction protocols to deal with high-starch tissues. The protocol from Wang et al. (2011) seems to work well enough in our hands, and we no longer have the insoluble pellet problem (although many of our samples are, unfortunately, fairly transcriptionally quiescent, so the yields aren't that high).

In summary, an insoluble, gel-like pellet at the end of an RNA extraction may be starch carry-over. A somewhat cloudy aqueous phase during the extraction (or, more extreme, a completely gelatinized aqueous phase) is a good indicator that you might run into this problem. The SDS and multiple extractions in the Wang et al. (2011) protocol seem to solve the problem.

No comments:

Post a Comment