problem in RNA extraction from mice liver and spleen - (Jun/19/2006 )

hello everbody

I am using RNA Bee for isolating RNA from mice liver and spleen. Within 2 min. after harvesting tissue we are homogenising the tissue in RNABee. But still we are getting degraded RNA (Not very intense band of 28S and 18S) . We are taking care in all steps like baking of scissors , forceps etc.
please help us. What about liquid nitrogen use?
thanks
Gurdeep

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It is quite difficult to isolate RNA from liver since there are so many proteases. I use twice the amount of Tissue lysing solution than waht is give in the protocol. Also, use test-tubes that are autoclaved (glass tubes work well for this purpose). You water has to be DEPC treated and autoclaved. And clean you work station with EtOH before use.

ALso, I add an additional extraction step (I use TRizol method and use an additional chloroform extraction before I precipitate the RNA).
Hope this helps
Good luck

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Sorry if this sounds stupid, but with the additional chloroform extraction do you mean that the same amount of chloroform initially added is added to the aqueous phase after it has been transferred to a fresh epp. tube? I'm assuming that this will improve both A260/280 and A260/230 ratios....right?

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