Analysis of Gene Expression in Striatal Tissue by Multiprobe RNase Protection Assay - Drugs of Abuse: Neurological Reviews and Protocols - page 184

Table 2
Tissue Weight and RNA Yield from Rat by Brain Region

Approximate Approximate yield ( µ g)

Tissue weight (mg) total RNA/animal

Caudate/ putamen 35 20

Nucleus accumbens 35 30

Substantia nigra/ventral tegmentum 30 20

Locus coeruleus 20 12

Prefrontal cortex 110 95

Hypothalamus 75 50

Olfactory tubercle 55 55

Hippocampus 75 63

Dissection was made by rodent coronal brain matrix (7) .

detect the transcript being measured. This will be determined by two variables:
transcript prevalence and mass of total RNA in the tissue studied. The weight
of tissue studied will determine the yield of total RNA extracted, which will
in turn guide details of the study being planned. We dissect brain tissue using
a coronal brain matrix, as has been described previously (7) . The mass of
individual brain section and total RNA yield obtained using this dissection
method are listed in Ta b l e 2 . We extract RNA according to the method of
Chomczynski and Sacchi (6) , modifi ed only slightly from (8) ( see Note 2 ).

1. Dissect tissue quickly on ice. Immediately freeze tissue in liquid nitrogen.
Homogenize individual tissue sample (weight 30-75 mg) in 810 µ L of denaturing

solution. Transfer to a 2-mL microfuge tube containing 81 µ L of 2 M sodium

acetate, pH 4.0.

2. Mix, add 810 µ L of water-saturated phenol, and mix.

3. Add 162 µ L of CHCl 3 -isoamyl EtOH (49 1), mix, and allow to stand on ice

for 15 min.

4. Centrifuge for 20 min at 10,000 g at 4°C.

5. Transfer the upper phase to a 2-mL microfuge tube containing 810 L of

isopropanol. Incubate for 30 min at -20°C. Centrifuge for 10 min at 10,000 g at
4°C, and resuspend pellet in 300

µ

µ L of denaturing solution.

6. Transfer to a 1.5- L of isopropanol. Incubate

for 30 min at -20°C. Centrifuge for 10 min at 10,000 g at 4°C.

µ L microfuge tube containing 300 µ

7. Wash pellet two times with 700 L of 75% EtOH. Vortex-mix, allow to stand

for 15 min at room temperature, and then precipitate by centrifugation for
5 min at 10,000 g .

µ

8. Air-dry the pellet, being careful not to overdry. Resuspend pellet in 100 µ L

of TE.