Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 160

5 min, and add 8.5 mL of 37% formaldehyde solution. Shake again and pour into
horizontal gel tray in chemical hood. Let cool.

2. Agarose: Low electroendosmosis (EEO).

3. Formaldehyde solution (37%).

4. Nylon membrane for nucleic acid blotting: Zeta Probe membrane (Bio-Rad) or
Genescreen membrane (Perkin-Elmer).

5. 50X TAE buffer: 242 g of Tris base, 57.1 mL of glacial acetic acid, and 18.6 g
of EDTA sodium salt in 1 L of distilled water.

2.3. Labeling of Probes
Many companies sell optimized kits that contain all necessary reagents for
labeling. When purchasing these kits, follow the instructions of the manufacturer.
In the following subheadings I provide generic protocols that can be used when
components are purchased individually. RNA polymerases are provided by the
manufacturer with optimized buffers that are used in the reactions.

2.3.1. Labeling of Riboprobes

1. 100 m M Dithiothreitol (DTT): Prepared in DEPC-treated distilled water. Stored
at -20°C.

2. Stock of rATP, rGTP, and rUTP: Purchase 10 m M stocks of all nucleotides in
DEPC-treated water or buffer, and mix one volume each of rATP, rGTP, and rUTP
and DEPC-treated distilled water (fi nal volume of each nucleotide: 2.5 m M ).
Store at -20°C.

3. RNasin or any other type of commercially available RNase inhibitor (e.g., from
Promega, Ambion, Invitrogen, Stratagene, etc.). Stored at -20°C.

4. RQ1 RNase-Free DNase (Promega). Stored at -20°C.

5. NucTrap push columns (Stratagene) or any other type of prepacked size-exclusion
chromatography column that retains nucleotides and short oligonucleotides to
separate them from larger pieces of DNA or RNA.

6. 1X STE buffer: 100 m M Sodium chloride; 20 m M Tris-HCl, pH 7.6; and 10 m M
EDTA.

2.3.2. Labeling of cDNA Probes

1. Stock of dATP, dGTP, and dTTP: Commercially available at 100 m M concentra-
tions of each nucleotide. Dilute one volume of dATP, one volume of dGTP, and
one volume of dTTP with one volume of distilled water to make a 25 m M stock
solution. Dilute 1 50 (500 µ M ). Store at -20°C.

2. Random oligonucleotide primers. Commercially available. Store at -20°C.

2.4. Hybridization of Probes to Nylon Membranes

1. 50X Denhardt's reagent: 1 g of Ficoll (type 400), 1 g of polyvinylpyrrolidone,
and 1 g of bovine serum albumin (BSA) (fraction V). Bring to 100 mL with
DEPC-treated distilled water. Store at -20°C.