Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos
Author(s)Ji, Ni; van Oudenaarden, Alexander
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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.
DepartmentMassachusetts Institute of Technology. Department of Biology; Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences; Massachusetts Institute of Technology. Department of Physics
Ji, Ni, and Alexander van Oudenaarden. “Single Molecule Fluorescent in Situ Hybridization (smFISH) of C. Elegans Worms and Embryos.” WormBook (2012): 1–16.
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