Quantitative In Situ Hybridization and Determination of mRNA Content

Abstract

The determination of mRNA levels by in situ hybridization in brain tissue is a prominent topic of concern of workers in this field. Two degrees of quantitation are theoretically possible. The ability to provide a minimum estimate of mRNA amounts for comparing conditions is the easier the two. The ability to measure absolute amounts of mRNA by in situ hybridization is a much less certain and more contentious proposition, as discussed below. In both cases, it is necessary to convert grain densities to tissue radioactivity to, finally, mRNA levels. The first conversion is discussed in the second portion of this chapter. Briefly, for isotopes with higher energy than tritium, emulsions of uniform thickness are required to eliminate variability in grain density due to variations in emulsion thickness. For this purpose, one can use x-ray film or emulsion-coated coverslips (1). In addition, one must simultaneously expose standards containing known amounts of the isotope used in the experimental sections (2).