Need reliable method for RNA extraction from lymphocytes

Howard T. Petrie h-petrie at ski.mskcc.org
Fri Oct 14 08:37:35 EST 1994


In article <37h5b1$ed3 at crocus.csv.warwick.ac.uk>, lsrbd at csv.warwick.ac.uk
(Chris Hodgson) wrote:

> Dear Bionetters,
> 
> I'm trying desperately to isolate quality RNA from lymph node cells
> (predominately lymphocytes) isolated from mice.
>

(heavily edited)

> with circa 10^7 cells each time :
> 
>


I have done RNA extractions for several years on sorted or whole
preparations of mouse thymus, lymph node and spleen.  Cell numbers rtange
from 250,000 to 10 meg per microfuge tube.  Integrity of ribosomal bands
after electrophoresis is always good.  Northern blots have been published
(Eur. J. Immunol. 20:2813;  J. Exp Med. 178:615; the latter has a photo of
EtBr staining of ribosomal bands).  Method is based on modifications of a
published manuscript (PNAS USA, 85:6082) and snippets from Molecular
Cloning:...  The whole thing takes about an hour (after preparation of the
cell suspension).  I don't use DEPC, just autoclaved water and other
solutions and I hold my breath a lot...

Start with single cell suspensions, however you would like to obtain
them.  To prevent clumping, I do all of my preps in HBSS/5% FBS including
10ug DNAse I/ml.  The latter IS NOT A PROBLEM for subsequent RNA or DNA
preparation (after the washes described below); I also prepare
megabase-sized genomic DNA from the nuclei isolated by this method.  The
DNAse is critical for preparing and maintaining good single cell
suspensions, especially during lengthy cell sorts.  All steps are on ice
until the Proteinase K digestion.  Dispense the appropriate number of
cells in the above medium into microfuge tubes (I don't perform any
special manipulation to these other than autoclaving); ten million cells
is a good upper limit for peripheral lymphoid cells, although I've done
twice this number for crude preps.  Centrifuge ca. 250 X g in a swinging
bucket rotor (your standard lab centrifuge with buckets for 12 X 75mm
tubes will hold microfuge tubes quite nicely).  Wash the cells once in
DPBS, without cations, and with 20mM EDTA in the same fashion.  Remove all
the supernatant using an aspirator and a drawn pasteur pipet.  At this
point, I usually flash spin in a microcentrifuge (not full speed) to get
the remaining fluid down (aspirate this) and to move the cell pellet to
the side, which facilitates the former.  If you have lots of cells to
spare and you're really worried about the DNAse, you can now repeat this
wash, but if you aspirate carefully, it's really not necessary.  Resuspend
the pellet without added solution, then add ca. 200 microliters of 150mM
NaCl/2mM MgCl2/10mM Tris pH8, which has been brought to 5% NP-40 and 20mM
vanadyl ribonucleoside complexes added immediately prior to use.  Vortex
10-15" and incubate on ice ca. 5' with occasional gentle vortexing.  If
you let this step go too long, you will start to see genomic DNA
contamination, but in my experience, it's still negligible after 10'. 
Spin at 4 degrees in a microfuge at maximum speed for 90".  Remove the
supernatant into a fresh tube which contains an equal volume (ca. 200uL)
of 300mM NaCl, 200mM Tris pH8, 25mM EDTA, 2% SDS, which has had Proteinase
K added to 100ug/ml immediately prior to use.  Mix by gently vortexing and
incubate for 30' at 37 degrees.  Add an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1), vortex to homogeneity (about
15"), and spin in a microfuge at maximum speed for 5' at room
temperature.  Remove the aqueous phase to a clean tube (note: if cell
numbers are limiting, you can back-extract the organic phase) and add 0.1
volumes of 3M NaOAc pH 5.6.  Mix and chill on ice before adding sufficient
absolute EtOH to bring the solution to 70% EtOH.  You may add 1ug of
glycogen at this point if you wish.  If I'm not using the RNA right away,
I store them at -70 at this stage; I've used them YEARS later with no
problems.  For use, spin, aspirate as much of the supernatant as possible
by flash spinning and using a fine-drawn pipet as described above; this
minimizes the time required to evaporate EtOH to a minimum (about 1-2'). 
Resuspend in TE and go for your life.  I didn't expect to write this much
detail, but now that I've done it, I hope you find it useful.



More information about the Immuno mailing list