RNA extraction from small initial tissue. Help!!

[Ksenia]

Dear all,

I am extracting RNA from flat worms (Gyrodactylus salaris) with Trizol-based method, but I do not get any RNA, or RNA amounts are very low (the control, fish tissue samples are extracted normally).

Alive worms were frozen in liquid nitrogen in a drop of water (40 microliters). The number of worms per sample is around 30, and they are quite small (0.1 miliimeters). From this fresh-frozen samples I get nothing, whereas I was able to extract RNA from the same number of RNA-later stored worms previously.

I tried the standard protocol; additionally homogenizing the frozen drop of water to powder with immediate adding of Trizol; letting the samples to sit longer in Trizol for better lyzation; elongating the RNA precipitation step, but nothing seems to work.

Does anybody have an experience with extracting from very small initial amounts of tissue or otherwise may suggest something?

I will appreciate your help!!