Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 163

2. After exposure of neurons to the drugs of interest, plates are removed from the
cell culture incubator, medium is quickly and completely aspirated, and plates
are immediately put on wet ice.

3. Optional: One or two washes with 1X PBS (1 mL each wash). We generally
do not wash.

4. For cell lysis, add 500 L of NP-40 lysis buffer to each well. Leave the cells

with lysis buffer on wet ice for 5 min for complete lysis, and then swirl plates
to lift the cells off. Resuspend cells by repeated pipetting up and down. Transfer
the cell solutions into Eppendorf tubes.

µ

5. To separate cell nuclei, centrifuge the tubes at 4°C for 5 min at 8000 g , then
transfer supernatants to fresh Eppendorf tubes, and discard pellets (cell nuclei
and membranes).

6. Add SDS to supernatants to a fi nal volume of 0.2% SDS (add 10 µ L of 10%

SDS). Optional: Samples can be frozen at this point.

7. For acidic phenol-chloroform-isoamyl alcohol extraction, bring up supernatants to
0.3 M sodium acetate (add 50 L of PCI (equal

volume), and invert tubes a couple of times and vortex-mix for 30 s. Centrifuge the
tubes for 10 min at 16,000 g , 4°C. Transfer upper phase (aqueous) to a fresh tube.
Do not touch the interphase; if you touch the interphase, repeat centrifugation.

µ L of 3 M sodium acetate), add 550 µ

8. For RNA precipitation, add two volumes of 100% ethanol to the aqueous phase
and leave the samples at -20°C for 30 min (minimum time: 5 min). Optional:
Samples in ethanol can be stored safely at -80°C for a prolonged time.

Centrifuge tubes for 10 min at 16,000 g at 4°C and carefully aspirate supernatants
and discard.

L of 75% ethanol to the pellets, vortex-mix the
pellets briefl y, and leave on ice for 10 min to dissolve residual sodium acetate.
Centrifuge tubes for 5 min at 16,000 g at 4°C and carefully remove supernatants
and discard.
10. Air-dry the pellets and take up in 12

9. To desalt RNA, add 500 µ

µ L of RNA gel running buffer.

11. Heat samples at 70°C for 10 min.
12. Place on wet ice.

We do not measure RNA content in these samples, and we use the entire
sample to run Northern blots; because all wells have approximately the same
number of cells, we have found very little loading differences in these gels.
The amount of RNA is at least 1

µ g/1 million neurons, 2-3 µ g per well of

a six-well plate.

3.2.2. RNA Extraction from Brain Tissue

This RNA preparation is modifi ed from ref. 4 , and can be used for up to
300 mg of tissue ( Fig. 5B ) ( see Note 3 ). We use this protocol for preparing
RNA from tissue (5 , 6) .