HELP: RNA isolation from cell cultures

Steven Poet poets at ccmail.orst.edu
Thu Aug 17 11:43:27 EST 1995


I second that.  We use Trizol LS from GibcoBRL to extract RNA from 
virus-infected cell lines and have had good results.  The key is
acidic phenol/chaotropic solution extraction to avoid DNA 
contamination in the aqueous layer.

"Dr. Angela Hofstra" <Hofstra at aa.wl.com> wrote:
>
> We routinely use Trizol from Life Technologies to harvest RNA from 
> cultured liver cells.  It works very well.
> 
> Angela
> 
> kazu at bio.hokudai.ac.jp (Suzuki Kazuhiko) wrote:
> >At 15:57 95.8.5 -0500, SEGALL,LAURA,MS wrote:
> >>Hi!
> >>I'm working in a lab that was recently set up, and we are having a
> >>very difficult time isolating RNA from cell cultures. We consistently
> >>get only about half of the expected amounts, and even then, we seem to
> >>have some DNA contamination since when we load say 5 ug RNA for a
> >>Northern, we see a much weaker band than we do for controls, so some-
> >>thing is interfering in our getting the proper concentration reading.
> >>We now spin the lysed cells for 45 min. rather than 20 min, and there
> >>seems to be some improvement, but we now think that there is something
> >>very seriously wrong with our Guanidine Thiocyanate solution (although
> >>we use 4M guanidine Hydrochloride with Na-citate and 10% Laurylsarcosyl?
> >>whatever, the detergent).
> >>We were told that the pH of the solution should be 7, but ours was at
> >>5.5. We made a new solution, and got the same thing. When we tried to
> >>fix the pH, we noticed that the citrate isn't really buffering the   lut
> >>solution at all. We've found detergent precipitates in it and were
> >>forced to heat it to clear it up a bit, although it didn't do much.
> >>At this point, any help is welcome.
> >>Thankyou in advance,
> >>Laurie.
> >
> >Dear Laurie
> > 
> >I have also been working in a lab similar to your circumstance
> > and faced to similar problem of RNA isolation from cell culture.
> >I have also taked place RNA isolation according to 
> >Guanidine Thiocyanate method from red book. 
> >I think you had better used GTC from Fluka, 
> >but I seem to take account for the pH of salt .
> >If you use sodium acetate, check pH that is  4.
> >As  pH is larger than 4, more DNA is contaminated in water layer 
> >that otherwise contain organic layer when phenol-chloroform-isoamyl extraction. 
> >In addition, I usually perform isopropanol precipitation in -20C overnight.  
> >Good luck.
> >
> >Kazuhiko SUZUKI
> >Graduate School of Science
> >Division of BIological Sciences 
> >Hokkaido University 
> >
> 
> 




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