Abstract
Gene expression is often regulated by the abundance, localization, and translation of mRNAs in both space and time. Being able to visualize mRNAs and protein products in single cells is critical to understand this regulatory process. The development of single-molecule RNA fluorescence in situ hybridization (smFISH) allows the detection of individual RNA molecules at the single-molecule and single-cell levels. When combined with immunofluorescence (IF), both mRNAs and proteins in individual cells can be analyzed simultaneously. However, a precise and streamlined quantification method for the smFISH and IF combined dataset is scarce, as existing workflows mostly focus on quantifying the smFISH data alone. Here we detail a method for performing sequential IF and smFISH in cultured cells (as described in Sepulveda et al., 2018 ) and the subsequent statistical analysis of the smFISH and IF data via three-dimensional (3D) reconstruction in a semi-automatic image processing workflow. Although our method is based on analyzing centrosomally enriched mRNAs and proteins, the workflow can be readily adapted for performing and analyzing smFISH and IF data in other biological contexts.