RNA from human hepatocytes in culture

[Coag]

I have attempted to extract RNA from human hepatoctytes (commercially available) which I have grown/maintained on collagen coated plates.

I am using the standard Trizol method which understood would work but am not getting any pellet, where am I going wrong? Should I be doing something extra to disolve the collagen.

Everything I've read says the standard protocol should work. The cells are expensive so I cant afford to mess around too much.
Any advice

[danfive]

How much tissue or cell count are you starting out with? It could be that your RNA pellet is too small to notice. Have you tried putting an aliquot in spectrophotometer & doing OD260/280? or some RT-PCR?

[admin]

Hello there Coag,
I have used Trizol and haven't had amazing success with it. I really like the column based kits ie Qiagen as they really do work well. The Trizol method really varies from lab to lab, and person to person so it could be any of the steps as well going wrong even if you got your amount right.
If you can get your hands on them, they really simplify things and get rid of most of the contaminating DNA, protein etc.

Cheers!

[Coag]

Thanks for the responses, admin we initially tried the Qiagen protocol and also got nothing. We came to the conclusion that the collagen was clogging up the column despite the proteinase K digestion step which is why we switched to Trizol since most of the papers on hepatocytes seem to use trizol.
On closer inspection I am wondering if our cell count was too low, so we are going to repeat it with rat hepatocytes (way cheaper) and use a glycogen carrier to see if that improves yield. If we get things to work we might have another go at the human hepatocytes.

[aquaplasmid]

Coag,

Would you like to try AquaRNA? Email [Only registered users see links. ] for a sample.