Neuropeptide gene transcription in central and peripheral nervous system tissue by nuclear run-on assay

Abstract

Rapid progress in molecular biological techniques has enabled the study of gene expression in a wide variety of neuro-endocrine tissues. There are two general methods for determining transcription of a specific messenger RNA (mRNA). In both methods, transcriptionally active nuclei are isolated from tissue, or cultured cells, incubated in the presence of labeled nucleoside triphosphate, and newly synthesized RNA is then isolated. Quantitation is then performed by the film autoradiography and the densitometric analysis of the autoradiographic band. This method has the advantages of permitting the investigation of transcription of several genes simultaneously and the increased sensitivity possible with prolonged exposure of the X-ray film. If hybridization does not proceed to completion, absolute counts are not accurate; thus, an inclusion of an internal standard––that is, a gene whose transcription does not change in response to the stimulus––is necessary to permit comparison among stimuli. The filter hybridization method is described with examples of its use in investigating neuropeptide gene transcription