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RNA impossible to dissolve


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#1 Flyingbike

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Posted 12 October 2009 - 01:54 AM

Hi everyone !

Last week I had some hepatocytes in primary culture. Some were transfected with siRNA using an adenoviral vector and the transfectant "PEI", others were just cultured with different effectors. I extracted RNA using the RNA plus reagent (works like trizol). Finally, I tried to dissolve pellets in water, as usual. The issue is that RNA form hepatocytes transfected as described before stayed as a pellet and did not dissolve at all, whereas others RNA did.

I don't have any idea of what could explain it. Here are solutions I tried :

-65 heating = no effect
-one more ethanol wash = no effect
-RNA quantification in "supernatant" = near zero but somethings absorbs at 230 nm (any idea ?)

What is going on in my tubes ?

Thanks a lot

#2 T cell

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Posted 12 October 2009 - 04:41 AM

Check the pH of your water, it might be to acidic.
Did you dry well your RNA pellet?

#3 Flyingbike

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Posted 12 October 2009 - 05:07 AM

Check the pH of your water, it might be to acidic.
Did you dry well your RNA pellet?


Hi and thanks for answering. Water used was the same in all the tubes, and some of them (without adenoviruses, siRNA and PEI) dissolved well. Drying time was also the same for all the samples, and the same i use everytime (speed-vac).

THe only difference was presence of adenoviruses, siRNA and PEI for a few hours (4h), then medium was changed for 24h, cells were washed in PBS and then RNA extraction was performed.

The ones with transfection are impossible to dissolve, the ones without, which I extracted in parallel, are already in solution.




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