Analysis of Gene Expression in Striatal Tissue by Multiprobe RNase Protection Assay - Drugs of Abuse: Neurological Reviews and Protocols - page 179

7. 5X Plasmid buffer: 250 m M Tris-HCl, pH 8.3, 375 m M KCl, and 15 m M MgCl 2 .
8. Dithiothreitol (DTT).
9. 10X Proteinase K buffer: 10 m M Tris-HCl, pH 7.8, 5 m M EDTA, 0.5 % sodium
dodecyl sulfate (SDS).
10. Proteinase K (Invitrogen) dissolved into 10 mg/mL solution, divided into 10- L

aliquots, and stored frozen at -20°C. It should not be freeze-thawed more than
fi ve times.

µ

2.2. mRNA Standard Production

1. Deoxyribonucleoside triphosphates (dNTPs): store at -70°C (they may be pur-
chased individually, or premixed as ribonucleoside triphosphate sets from Roche).

2. Diethyl pyrocarbonate (DEPC)-treated water (5) .

2.3. Radiolabeled cRNA Probe Preparation
Probes may be synthesized as described in Subheading 3.3. using the
following individual reagents.

1. DNA-dependent RNA polymerases (SP6, T7; Promega).

2. RNasin (Promega).

3. RQ1 DNase (Promega).

4. Nuctrap columns (Stratagene)

5. [ - 32 P]UTP (800 Ci/mmol; New England Nuclear).

Alternatively, commercial kits are available containing nonradioactive reagents
required for probe preparation, such as MAXI script kit (Ambion, Austin, TX).

2.4. mRNA Sample Preparation
Commercial kits containing needed reagents, and directions for their use,
are available (TRI REAGENT, Molecular Research Center, Cincinnati, OH).
Alternatively, the following recipe may be prepared.

1. Stock denaturing solution (100 mL): 50 g of guanidine isothiocyanate (GIBCO,
cat. no. 5535UA), 59.48 mL of H 2 O, 2.64 mL of 1 M sodium citrate, pH 7, and
5.28 mL of 10% sarcosyl, heat at 62.5°C to dissolve. Store at room temperature
up to 3 mo.

2. Working denaturing solution: Add 350 -mercaptoethanol and one drop

of antifoam A (Sigma) to 50 mL of stock denaturing solution. Store refrigerated
up to 1 mo.

µ L of

3. Coronal brain matrix for brain tissue dissection (Research Instruments and
Manufacturing, Corvalis, OR).

2.5. Ribonuclease Protection Assay
The assay can be performed using individual reagents including RNase ONE
(Promega). Alternatively, commercial RNase protection assay kits such as RPA II
Kit (Ambion) include all reagents required for assay.