Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 174

too short or the wrong polymerase was used. To solve the problem, use RNase-
free working area and tools, and precipitate template with 75% ethanol at -20°C
for 20 min, centrifuge for 10 min at 16,000 g at 4°C, aspirate supernatant, wash
pellet with 1 mL of 75% ethanol, centrifuge for 5 min at 16,000 g at 4°C, aspirate
the supernatant, and let the pellet air-dry. This procedure should remove residual
salts from the template. You may have to redetermine the amount of DNA. Use
all solutions at room temperature, do not use ice, and make sure the restriction
enzyme used for cutting the vector leaves at least a 300-basepair sequence from
the polymerase site.
11. For the purpose of reprobing the membrane, cDNA probes can be stripped off
by adding boiling 0.1% SDS in distilled DEPC water, then letting it come down
to room temperature while shaking for 30 min. Riboprobes cannot be stripped.
These blots can be reused only after the radioactivity is decayed.
12. If the DNA template is in low melting point agarose, the agarose may inhibit
the labeling reaction. It is also important to stay at room temperature or higher
during the labeling to avoid gelling of agarose. Extract DNA template from the
agarose to minimize this problem.
13. The sticky probe may make X-ray fi lm come out too dark. Rewashing the blot
may solve this problem.
14. Visible ribosomal band is a problem if the specifi c band is too close to the
ribosomal bands. Decreasing the amount of radiolabeled probe added to the
hybridization solution may solve the problem.
References
1. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual , 2nd edit. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.
2. Rajadhyaksha, A., Barczak, A., Macías, W., Leveque, J. C., Lewis, S., and
Konradi, C. (1999) L-type Ca 2+ channels are essential for glutamate-mediated
CREB phosphorylation and c-fos gene expression in striatal neurons. J. Neurosci.
19, 6348-6359.
3. Perry, R. P., La Torre, J., Kelley, D. E., and Greenberg, J. R. (1972) On the lability
of poly(A) sequences during extraction of messenger RNA from polyribosomes.
Biochim. Biophys. Acta 262, 220-226.
4. Berger, S. L. and Chirgwin, J. M. (1989) Isolation of RNA. Methods Enzymol.
180, 3-13.
5. Leveque, J. C., Macías, W., Rajadhyaksha, A., et al. (2000) Intracellular modulation
of NMDA receptor function by antipsychotic drugs. J. Neurosci. 20, 4011-4020.
6. Konradi, C., Leveque, J. C., and Hyman, S. E. (1996) Amphetamine and dopamine-
induced immediate early gene expression in striatal neurons depends on postsyn-
aptic NMDA receptors and calcium. J. Neurosci. 16, 4231-4239.
7. Feinberg, A. P. and Vogelstein, B. (1983) A technique for radiolabeling DNA
restriction endonuclease fragments to high specifi c activity. Analyt. Biochem.
132, 6-13.