I’m looking for a protocol to isolate total RNA from a small sample for qPCR…from say a sample size of ~30-50 worms. Any suggestions?
I think you can make a collection of several small-sized samples at -80 ℃. And I help this protocol can help you.
THat’s great…thanks!
Precipitation of small amounts of RNA can be problematic, and glycogen can affect the RT reaction. We’ve had good success using linear polyacrylamide (GenElute LPA from Sigma) as a carrier (protocol from Krause lab).
1-Pick 25 worms into 30 ul aqueous buffer (e.g., TE).
2-Add 300 ul Trizol and mix thoroughly.
3-Add 5 ug LPA; vortex 10 seconds.
4-Add 60 ul chloroform; vortex 30 seconds.
5-Spin 5 minutes @ 14K RCF.
6-Transfer aqueous phase to new tube.
7-Add 0.8 volumes isopropanol.
8-Precipitate overnight @ -20.
9-Spin 20 minutes @ 14K RCF.
10-Carefully aspirate supernatant via pipette.
11-Wash once w/ 500 ul 75% ethanol.
12-Spin 10 minutes @ 14K RCF.
13-Carefully aspirate supernatant via pipette.
14-Air-dry 1-2 minutes.
15-Resuspend in appropriate volume RNase-free water.
Good luck,
Harold
Thanks for the suggestion. Really nice timiing actually…I just tried the first protocol, and yes, there wasn’t really a pellet. I’ll will definitely try the GenElute LPA. Thanks.
same things happened here, the first method mentioned did not worked well in my lab as well. :-[
just wonder how did they manage to isolate from 10 worms ???
The GENELUTE method works really well 
I isolated RNA from 30 worms using it.