In situ hybridization: Quantitation using radiolabeled hybridization probes

Abstract

This chapter describes several approaches for the determination of relative cellular hybridization densities using in situ hybridization. In other circumstances that might wish to determine the absolute cellular content of messenger RNA (mRNA), the accurate determination of this second quantity may be more difficult without the use of additional adjunctive studies of extracted RNAs. In situ hybridization techniques are finding increasing use when the localization of gene expression is of importance and, especially when regulated gene expression is studied in heterogeneous cell populations. Recent reviews document approaches for localizing specific nucleic acid sequences to particular cell populations by in situ hybridization. As approaches to in situ hybridization mature, and as more questions arise about gene regulation in individual cells, quantitative in situ hybridization analyses will assume increasing importance. With careful attention to specific technical details, this approach should yield data that can help to address many of these biological questions.