Need reliable method for RNA extraction from lymphocytes

Chris Hodgson lsrbd at csv.warwick.ac.uk
Wed Oct 12 12:10:57 EST 1994


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



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