Detection of mRNA by In Situ Hybridization and RT-PCR

Abstract

This chapter provides an overview of several approaches currently being used that rely on messenger RNA (mRNA) from a relatively small amount of starting material as a means to obtain detailed information on the expression of a single gene or an entire network of genes in the embryo. Whole mount in situ hybridization (WMISH) is a relatively easy procedure, resulting in specimens with long-term stability and great utility for deciphering gene expression patterns. The WMISH protocol presented is optimized for preserving specimen morphology and handling small numbers of specimens. In situ hybridization methods use sectioned tissue and radioactively labeled RNA probes. It provides a more quantitative assessment of relative mRNA target levels and also offers a good way to compare the distributions of different mRNAs in the same embryo by probing adjacent thin (1 μm) sections. In situ signals for a large number of different mRNAs as a function of developmental stage are always consistent with other hybridization assays, such as RNA blotting or RNase protection. Whole-mount in situ hybridization largely replaces radioactive methods with sectioned tissue because of its relative speed, the long-term stability of the probes, and the ease of three-dimensional interpretation. The detection of mRNA by real time quantitative PCR (RTQ–PCR) helps to obtain accurate relative quantity of mRNA expression for as many genes as possible in direct comparisons between differentially manipulated experimental samples using relatively small quantities of starting material.