Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 162

rinsing with DEPC-treated distilled water. One of the latter three procedures
should also be used to decontaminate electrophoresis tanks. Do not use DEPC in
polycarbonate or polystyrene containers.

4. Label chemicals used for RNA work and keep separate from common chemical
stocks. Always use gloves when handling these chemicals.

5. Decontaminate solutions by adding 1 1000 volume of DEPC, stir for a couple of

hours, and autoclave for 1 h to hydrolyze/inactivate DEPC. Solutions containing
Tris react with DEPC and cannot be decontaminated with DEPC. For these
solutions, DEPC-treated distilled water (autoclaved after treatment) should be
used. Solutions with thermolabile substances should be prepared with DEPC-
treated distilled water and fi ltered through a 0.2- m fi lter (e.g., syringe fi lter).

DEPC destroys and should not be brought into contact with polycarbonate or
polystyrene.

µ

3.2. RNA Extraction
I present two different methods that we have used successfully and consis-
tently in our laboratory, one for primary neuronal culture ( see Fig. 5A ), and one
for brain tissue ( see Fig. 5B ). The difference in the preparations are (1) in the
number of samples that can be worked up simultaneously, (2) in the speed of
separation between RNA and RNases, (3) in the strength of RNase-denaturing
conditions ( see Note 1 ), and (4) the maximum amount of tissue that can be
used in the preparation.

3.2.1. RNA Extraction from Primary Neuronal Culture

This RNA extraction is limited to small numbers of neurons or cells that
do not have high levels of RNases. The RNase-denaturing conditions in this
protocol are very gentle, and a quick separation of RNA from RNases is
essential ( see Note 1 ). All procedures need to be performed on ice at 4°C. Only
cells in a dissociated state, such as they are in culture, should be used, as this
protocol does not involve dissociation of tissue. We have used this protocol
with good results for primary striatal culture ( Fig. 5A ), primary cortical culture,
primary hippocampal culture, and cerebellar granule neurons (2) . In the fi rst
step of the protocol, nuclei with DNA must be separated from cytoplasm to
keep contamination of the RNA preparation with DNA at a minimum. Acid
extraction with phenol-chloroform-isoamyl alcohol (PCI) is used to extract
RNA into the aqueous phase, while DNA and proteins partition into the organic
phase or interphase (3) ( see Note 2 ). RNA is then precipitated and cleaned
from salt contaminants.

1. Primary neurons are grown on six-well plates for at least 1 wk, and each well
has between 2 and 3 million neurons.