Oligonucleotide

Gene expression

Gene

Confocal

Fluorescence in situ hybridization

Cell biology

Mathematics

In situ hybridization

Biology

Messenger RNA

Confocal microscopy

Chromosome

Genetics

Molecular biology

Geometry

Nature Protocols

Single-molecule fluorescence in situ hybridization (smFISH) enables mRNA quantification while preserving spatial information. Trcek et al. describe an smFISH procedure for Drosophila embryos, using wide-field, confocal and structured illumination miscroscopy. Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured i...

mRNA quantification using single-molecule FISH in Drosophila embryos