Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 158

effectively. The methods used for RNA extraction should be chosen according
to the RNase content of the tissue. A preparation that may work for one type of
tissue may not work for another. Pancreatic tissue is particularly well known
for high content of RNases (e.g., RNase A), while brain tissue seems to be less
vulnerable to RNA degradation. Because the preparations that are the most
conservative can also be the most time consuming and expensive, it is advised
to try out different preparations.
RNases are not only in tissue, but also on the skin and in saliva. A common
source of RNA degradation is contamination with RNases from the skin of the
investigator. It is therefore imperative that gloves are worn when handling RNA
or any type of chemicals and containers that will be used for RNA extractions.
Because the fi rst steps of RNA extraction are usually performed under RNase-
denaturing conditions, reintroduction of RNases from outside sources is most
detrimental in the latter stages of RNA preparation, when RNA is at its purest
and least protected.

2. Materials
For common molecular biology solutions see also ref. 1 .

2.1. RNA Extraction
2.1.1. RNA Extraction from Primary Neuronal Culture

Important: All buffers and stock solutions are treated with diethyl pyrocarbonate
(DEPC) and autoclaved, or brought to fi nal volume in DEPC-treated distilled
water. Tris buffer is prepared with DEPC-treated distilled water.

1. Phosphate-buffered saline (PBS) (for optional washing of cells): 11.5 g of
Na 2 HPO 4 , 3.0 g of NaH 2 PO 4 •2H 2 O, and 5.84 g of NaCl. Bring up to 1 L with
distilled water, adjust pH to 7.4-7.6, and add 1 mL of DEPC. Stir for at least
1 h, and autoclave.

2. Tris-HCl buffer stock solution: 1 M Tris-HCl, pH 7.6. Bring Tris up to three
fourths of fi nal volume with DEPC-treated distilled water; adjust pH to 7.6 with
1 N HCl, and bring up to fi nal volume with DEPC-treated distilled water. Note :
Rinse pH meter with DEPC-treated distilled water before use.

3. Nonidet P-40 (NP-40) lysis buffer: 50 m M Tris-HCl, pH 7.6, 100 m M NaCl, 5 m M
MgCl 2 ; and 0.5% (v/v) NP-40. Prepare with DEPC-treated distilled water.

4. 10% Sodium dodecyl sulfate (SDS): 1 g of SDS; bring to 10 mL with DEPC-
treated distilled water.

5. 3 M Sodium acetate, pH 4.5: pH sodium acetate with acetic acid to pH 4.5.
Prepare with DEPC-treated distilled water.

6. Phenol-chloroform-isoamyl alcohol (PCI): 25 mL of phenol, 24 mL of chloro-
form, and 1 mL of isoamyl alcohol. Note : Phenol needs to be saturated with
distilled water before it is added to the mixture ( see item 7 ). Store at 4°C.