RNA extraction from polysomes

[ngerges]

Hi everyone
I hope someone on here can help as I am going crazy..

I want to run a microarray analysis on polysomes ..

What I do is grow my human cell lines, extract the polysomes using a particular buffer, run the polysomes on a sucrose gradient, fractionate the gradient, and then try to extract RNA from the gradients for the microarray.

I am having LOTS of trouble with the RNA extraction.
1. I've tried 2 QIAGEN kits, and the Invitrogen Trizol protocol. I am finally able to get more than 200ng/uL and a 260/280 of about 1.6-2. BUT my 260/230 is ALWAYS below 1 which means I have TRIZOL contamination. Any suggestions on another protocol to extract my RNA. I usually add 2.5X EtOH to my samples overnight -80C before beginning any of the protocols...

2. When I run my samples on a bioanalyzer to check for quality, I don't get a smear.. nor do I get the 2 distinct bands. I get more than 30 random bands EVERYWHERE. I don't know what that means!

3. When I try to run my sample on an agarose gel instead, either (a) my sample floats out of the wells (I think I have EtOH in my final sample) or (B) I am able to run the samples by tripling the amount of loading dye but I see absolutely nothing on the gels.. As if there is no RNA...but that's not possible because the nanodrop points to the fact that I do.

Sorry this is so long but I've tried everything and am starting to lose it. I don't know what else to do. Any help will be much appreciated!

Thanks
NG

[bhardwar]

Hi NG,
Hope you are done with ur experimets. I am exactly facing the same problem as u mentioned. Any suggestion for improvement..
Thanks
RB