This chapter describes a run-on transcription assay that measures the number of RNA polymerase complexes transcribing a specific gene and an S1 nuclease protection assay that can be used to quantitate the absolute amount of either primary transcript or mature messenger RNA (mRNA). The protocols discussed in this chapter are designed to enable one to study possible hormonal regulation of neuro-endocrine peptide genes at each of these steps of mRNA biosynthesis. Transcriptional analysis of neuro-endocrine genes can be performed with flesh tissue or primary cell or clonal cell line cultures. The basis of the assay is to treat either the animal in vivo or the culture in vitro with the desired hormone for a specified time and then to isolate cell nuclei by homogenizing the tissue in a cold buffer containing Triton X-100, which solubilizes the cell membrane and allows for the nuclei to be pelleted at a low-speed centrifugation.
ASJC Scopus subject areas
- Molecular Biology