My lab has been having trouble isolating daf-2 RNA for qtPCR using a Qiagen Absolutely RNA kit. Can anyone offer suggestions about how to improve isolation using this kit or some other method?
My guess is that you might have already solved your problem in RNA isolation.
I just back to work with worm ; My suggestion is to combine the Trizol lysis first (plus frozen-thawn 3x)
and column use from QIAgen. Best of luck with your experiments.
My lab is using a tissue lysis procedure that involves tissue homogenization using a tissuelyser. I have a paper that I could give to you if you are interested.
If you are extracting RNA form a population of 200 worms or more: Suspend worms in 1mL Trizol. Freeze at -80C. Thaw. Extract once with Chloroform. Isopropanol precipitate . 70% EtOH wash. Resuspend in DEPC-water.
I follow that same protocol, but found that freeze-thawing the trizol-treated worms 6 times in liquid nitrogen greatly increases yield over freezing it to only -80˚C.