problem with degradation of cytoplasmic RNA fraction when making cytoplasmic and nuclear fractionated RNA

[Sharon Cooperman]

Hi,

I am trying to make fractionated cytoplasmic and nuclear RNA from 
mouse bone marrow and MEL (erythroleukemia) cells.  I have tried the 
Qiagen alternate protocol (on their web site) and the Norgen Biotek 
nuclear/cytoplasmic RNA fractionation kit.  My nuclear RNA looks 
great, but my cytoplasmic RNA looks very degraded (no ribosomal 
bands).  I know that there is little protection from RNase during the 
cytoplasmic lysis/nuclear spin down steps of the protocol and I 
wonder if I'm getting degradation of my cytoplasmic RNA during those 
steps of the protocol.  On the Norgen web site they show beautiful 
photos of undegraded ribosomal bands of cytoplasmic RNA made using 
their kit, but they are using HeLa cells and I wonder if bone marrow 
has more RNase than HeLa cells and that's why my result is not as 
good.  Does anyone have any experience isolating cytoplasmic RNA and 
any suggestions for me (such as a better protocol, a way to inhibit 
RNase, what RNase inhibitors to use, etc)?  Any advice or 
encouragement will be greatly appreciated!

Thanks!
Sharon
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Sharon Cooperman        	     <scoop from mail.nih.gov>
NIH, NICHD, CBMB                     301.741-6092
Building 18T, room 101               301.402-0078 fax
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