We do isolation from human lymphocytes all the the time. We use the
"everybody uses it Chomzynski method". I will assume that your cells are
relatively intact, therefore one can only assume that you have a bad
solution or that the RNA is degraded following isolation. This method is
relatively bulletproof because the RNA is in guanidinium during most of
the procedure EXCEPT for the last steps. Have you checked the quality of
the ethanol/isopropanol that you use for precipitation? At this point,
the RNA is *not* protected and can be degraded if your ETHOH is not
clean.
As an additional note, if you don't care how much tissue you are using,
you can lyse the entire node in guanidinium without bothering to
dissociate the cells. I just vortex the tissue peice in the guanidinium
(solution D by Chomcynski) for several minutes.
Good luck.
==============================================================================
James F. George, Ph.D. "Back off man, I'm a scientist"
Department of Surgery --Bill Murray
University of Alabama at Birmingham
205-934-4261 voice
txpljfg at uabcvsr.cvsr.uab.edu
===============================================================================
On 12 Oct 1994, Chris Hodgson wrote:
> Dear Bionetters,
>> I'm trying desperately to isolate quality RNA from lymph node cells
> (predominately lymphocytes) isolated from mice.
>> I've kept the nodes on ice up until I push out the cells through gauze then
> wash pellets with ice cold PBS. This applies to only two of the methods used
>> I've tried the following with circa 10^7 cells each time :
>> - Detergent (NP40) lysis method from Sambrook & Maniatis : yields low,
> quality poor (no intact ribosomal RNA bands)
>> - RNA -ALL method - was posted to this newsgroup in July. Totally useless,
> yields nothing at all on three attempts, even with CHO cell lines !
>> - Chomzynkis "everybody uses it" method : Often quoted and tried, always
> gives degraded or DNA only
>> - Guanidinium / CsCl protocol / as in Maniatis : Tried this twice now
> getting nothing or very little which is degraded.
>>> I always use DEPC treated water (exposed myself to enough for a lifetime in
> two years !) and take care to use eppendorf tubes fresh from manufacturers
> clean packaging (wet-autoclaving can expose plastics to RNases). I shear
> samples in GITC buffer to ensure that no large weight DNA can cause problems
> through its viscosity.
>> I'm really struggling here - can anyone help me out ? Can anyone in the UK
> doing RNA extractions for lymphocytes spare me a few hours one day to show
> me how they do this ? I don't mean from cloned B-cells or T-cells as these
> usually have good levels of stable RNA for isolation.
>> Maybe you know of someone doing such isolations from lymphocytes routinely -
> if you do, show them this.
>> Cheers guys and gals
>> Chris
>> --
> Chris Hodgson \Where all roads lead to mystery
> University of Warwick \Serendipity will be found
> Coventry, U.K. \--<MiCrObE..MaNiAc>-94--
> +44 203 523561 \lsrbd at csv.warwick.ac.uk>>
