Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 161

2. 5X Buffer P: 5 M Sodium chloride, 250 m M Tris-HCl buffer, pH 7.4-7.6; and
0.5% sodium pyrophosphate. Prepare with DEPC-treated distilled water.

3. 50% Dextran sulfate: Bring 50 g of dextran sulfate to 100 mL with DEPC-treated
distilled water, shake vigorously overnight at 4°C in a shaker, and make sure it
is completely dissolved. Store at -20°C.

4. 20% SDS: 20 g of SDS, bring to 100 mL with DEPC-treated distilled water.
Caution: Wear a mask.

5. Salmon sperm DNA: 10 mg of salmon sperm DNA/mL of DEPC-treated distilled
water. Autoclave for 30 min, shear by pushing rapidly through a 20-gauge syringe
20 times, and store at -20°C.

6. Hybridization solution: 5 mL of 100% formamide, 2 mL of 50X Denhardt's
reagent, 2 mL of 5X buffer P, 2 mL of 50% dextran sulfate, and 0.5 mL of 20%
SDS. Add 100 L of boiling salmon sperm DNA to hybridization bottle (fi nal

concentration: 100

µ

µ g/mL).

7. 20X Saline sodium citrate (SSC), pH 7.0: 175.3 g of sodium chloride and 88.2 g
of sodium citrate. Bring up to 900 mL with distilled water, and measure the pH (it
should be around 7.0). Bring up to 1 L; this solution does not need to be RNase
free, as RNA bound specifi cally to complementary RNA (double-stranded RNA)
is not subjected to degradation by RNases.

3. Methods
3.1. Setting Up the RNA Workplace
RNA is very susceptible to hydrolysis by RNases. RNases are present not
only in tissues, but on the skin of investigators, and can thus be reintroduced
into the preparation at any time. RNases are very diffi cult to inactivate. They
are resistant to metal chelating agents, are active over a wide pH range, and
can survive prolonged boiling or autoclaving. Indeed, if autoclaved reagents
contain microorganisms, their RNases can be set free during autoclaving.
Fortunately, RNases rely on histidine residues for catalytic activity and they
can be inactivated by the alkylating agent DEPC, by 3% hydrogen peroxide
solutions, 1% SDS solutions, or 1 N sodium hydroxide solutions. Glassware can
also be baked at 180°C for several hours. The following steps should be
taken before starting to work with RNA:

1. Always use gloves when working with RNA. If you need to communicate during
the assay, use a face mask to avoid saliva contamination of your preparation.

2. Try to maintain a separate work area for RNA work, with its own set of RNase-
free labware and pipets. Always use gloves in this area.

3. Use sterile, disposable plasticware and bake your glassware in an oven at
180°C for 3 h or longer. Many companies guarantee that their plasticware is
RNase/DNase free. RNases can also be destroyed by soaking labware in 3%
hydrogen peroxide solution, 1% SDS, or 1 N sodium hydroxide solution, and