Estimation of tissue mRNAs by in situ hybridization

Abstract

We describe a new stereological method for analyzing data derived from the in situ hybridization procedure. This method should prove important, since data summarization in terms of grains per anatomic area by sampling of tissue sections may lead to faulty interpretations. Using computer simulation of measurements taken from a two- and a three-dimensional perspective, we show how the detection of molecular changes can be influenced by multiple structural events. Cell volume, the volume of the structure, and the number of labeled cells in an anatomic structure are all important parameters that must be assessed to obtain accurate results. We then outline in detail a simple and efficient method for estimation of the number of mRNA molecules in cells and in the total structure. By estimation of the abundance of mRNA molecules in three dimensions, we can make better inferences concerning gene expression in a structural context.