Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos

Abstract

In C. elegans, the expression pattern of a gene provides important clues to understanding its biological function. To accurately depict endogenous transcriptional activity, a highly sensitive method is required to measure transcript levels in the intact tissue across various developmental stages. Conventional RNA in situ hybridization methods using hapten- (biotin or digoxygenin) labeled RNA probes rely on antibody binding for visualization, and are thus only semi-quantitative at best (Raap et al. 1995; Levsky et al. 2003). Additionally, hapten-labeled probes are prone to diffuse localization (when conjugated with alkaline phosphatase), low sensitivity (when conjugated with fluorescent molecules), and non-specific probe binding. Here, we introduce a recently developed mRNA in situ hybridization method (Raj et al. 2008) that circumvents the above difficulties to give single molecule resolution of transcript detection