HELP: RNA isolation from cell cultures
Dr. Angela Hofstra
Hofstra at aa.wl.com
Thu Aug 17 09:11:27 EST 1995
We routinely use Trizol from Life Technologies to harvest RNA from
cultured liver cells. It works very well.
kazu at bio.hokudai.ac.jp (Suzuki Kazuhiko) wrote:
>At 15:57 95.8.5 -0500, SEGALL,LAURA,MS wrote:
>>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,
>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.
>Graduate School of Science
>Division of BIological Sciences
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