In one of our projects we want to extract RNA from yeast for measurements using the nifty NanoString nCounter technology. One of the nice things about this assay is that it uses very small amounts of actual material to measure RNA quantities. Moreover, unlike other assays, we do not need to purify RNA before the assay, which saves a lot of headaches.
The challenge we posed to Assaf, who is about to do the experiment, is how to extract cellular material from a very small yeast sample. We actually need as few as 15,000 cells (just for reference, 1 microliter of happy growing yeast has about 1-2 x 10^7 cells). The problem is that yeast, like many other microorganisms, has a strong cell wall made from proteins.
The classical way to break it is using mechanical action -- beating the yeast using small glass beads. This technique, however, does not scale down for small samples. And so, we decided to go with Plan B. Here we use an enzyme, Zymolase, that digests the cell wall. This creates spheroplasts -- cells without cell walls that are encapsulated by a thin membrane. We can then pop these open by diluting the sample with water with small amount of detergent. This solution also includes protein denaturing agents that block any RNAses from breaking down the fragile RNA, and thus preserves our sample.
Assaf and Ayelet tried this technique, and it seems successful. The zymolase readily digested the cell wall and left with nice spheroplasts. Then when he dilluted these quickly disappeared. Assaf made a nice movie of this process under the microscope. What you see is a small drop with yeast spheroplasts, and midway through the movie this drop is dilluted.
No comments:
Post a Comment