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

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
--
Sharon Cooperman <[Only registered users see links. ].gov>
NIH, NICHD, CBMB 301.741-6092
Building 18T, room 101 301.402-0078 fax
Bethesda, MD 20892