I've been extracting RNA from my HUVEC cells, do they look ok? I'm familiar with fungal RNA, but the HUVEC RNA doesn't look quite like I expected. The 28S band doesn't seem like it is 2x intensity of the 18S band. I used the Qiagen RNeasy Minikit, used the buffer RLT + B-mercaptoethanol added to monolayer and pipetted several times. Then pipetted into microfuge tube and vortexed 30sec. Then continued with the rest of the kit protocol.
I've also tried Trizol, yield is higher but protocol takes more time so I prefer the Qiagen kit. Is there anything I've done wrong? Only I haven't done DNase digestion, plan to do that today and run another gel and see.
First picture's Trizol has nothing, I don't know what happened there. 2nd pix is another batch of HUVEC RNA + Candida RNA run on fornmaldehyde gel. 3rd pix is the same RNA run on normal agarose gel.
All RNA reverse transcribed and PCR with b-actin gave correct bands.
I use 5x104 cells per well.
Edited by chrisbelle, 14 February 2009 - 09:02 PM.