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
