Quantification of mRNA in Neuronal Tissue by Northern Analysis - Drugs of Abuse: Neurological Reviews and Protocols - page 171

Fig. 5. Northern blots with the two RNA extraction methods described. (A) RNA
from a primary striatal culture treated with glutamate. NA was prepared with the
extraction protocol for primary neuronal culture. The membrane was developed with
a c- fos riboprobe, stored dry to let the radioactivity decay, and subsequently probed with
a cyclophilin random hexamer labeled cDNA probe. Cyclophilin is used to demonstrate
equal loading (11) . (B) RNA from rat striatum prepared with the CsCl ultracentrifuga-
tion protocol. Rats were treated with either saline control or amphetamine. The
membrane was developed with a c- fos riboprobe, stored dry to let the radioactivity
decay, and subsequently probed with a cyclophilin random hexamer labeled cDNA
probe.
9. Wrap wet membrane in saran wrap and expose to X-ray fi lm with intensifying
screens at -80°C ( see Notes 13 and 14 ). Alternatively, if you have access to a
phosphorimager system, use this system
10. Representative Northern blots for primary striatal culture and rat striatum are
shown in Fig. 5 .
4. Notes
Following is a list of problems that may be encountered, with suggestions
on how to uncover and correct these problems and how to avoid them in
subsequent preparations.
1. RNA is degraded by RNases from brain tissue and external sources. Tissue needs
to be frozen immediately after harvest at -80°C and should not be subjected to
freeze-thaw cycles. Primary neurons from culture cannot be frozen before nuclei
have been separated. These cells need to be harvested rapidly in lysis buffer on
wet ice, centrifuged in the cold room, and supernatants can then be frozen or
worked up further. RNases need to be inactivated rapidly by denaturing solutions
(e.g., guanidine thiocyanate) and separated from RNA at low temperature (4°C).