Methodology Used to Study the Influence of Thyroid Hormone on Regional Brain Gene Eexpression

The study of gene expression in the CNS requires techniques that allow regional and cellular resolution. To examine the regional and cellular distribution of specific mRNAs, in situ hybridization should be used. This technique is based on the hybridization of a labeled probe to the mRNA present in the tissue. The probe can be either DNA or RNA. DNA probes can be prepared from cDNA or from oligonucleotides. The highest specificity and signal-to-noise ratios are achieved with RNA probes, or riboprobes. These are complementary RNAs synthesized in vitro with phage RNA polymerases using as template the specific cDNA. Depending on the position of the polymerase promoter relative to the cDNA sequence, sense or antisense riboprobes can be synthesized. The antisense RNA will hybridize to tissue mRNA, whereas the sense RNA will not and is used as a control of the hybridization. There are a number of techniques to label the probes using either isotopic or nonisotopic methods (for descriptions of different methods and applications, see refs. 27-29). Among the nonisotopic, digoxygenin or biotinylated probes are the most commonly used. In our experience, the use of radioactive probes, in general, provides more sensitivity and specificity than nonisotopic probes, although the latter allows the visualization of cell morphology. Radioactive probes also allow for quantification of the signal. If a nonisotopic method is used for simplicity, convenience, or to avoid radioactive waste, it is advisable to compare the hybridization pattern with that obtained using an isotopic probe.

In this chapter, we describe an isotopic in situ hybridization method which is currently used in our laboratory. Sections from the rat brain are hybridized in flotation with [35S]UTP-labeled riboprobes. The use of floating sections results in better signal-to-noise ratios than hybridizing the sections previously immobilized on glass slides, although the analysis of fragile and/or small sections, for example those obtained from embryonic tissue, is more difficult. After hybridization, the signal can be detected by autoradiography, using X-ray films, or photographic emulsions. Autoradiography using X-ray films provides a regional pattern of expression of the target. Emulsion autoradiography is used when cellular resolution is desired. If combined with immunohistochemistry, the cells expressing the gene of interest can be identified using specific antibodies.

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