Quantitative single-molecule mRNA fluorescent in situ hybridization in C. elegans

Abstract

One of the key steps in determining the biological function of a gene of interest is to examine its expression pattern. Traditionally, gene expression in C. elegans has mostly been studied through reporter transgenes in which upstream regulatory sequence is fused to marker genes such as green fluorescent protein (GFP) or lacZ. In addition, expression can be determined through RNA in situ hybridization or antibody staining. An important downside of these different approaches is that gene expression is not measured quantitatively. Here, we describe a technique termed single-molecule mRNA fluorescent in situ hybridization (smFISH) that visualizes endogenous mRNA transcripts of the gene of interest at single-molecule resolution. These smFISH spots can be computationally counted to provide a highly quantitative measure of gene expression in vivo.