HELP: RNA isolation from cell cultures

Suzuki Kazuhiko kazu at bio.hokudai.ac.jp
Wed Aug 16 23:06:31 EST 1995


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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