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.
Highlights
[Background] single-molecule RNA fluorescence in situ hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple short fluorescently-labeled DNA oligonucleotides (“probes”) complementary to the target RNA (Femino et al, 1998; Raj et al, 2008). In this technique, when an ensemble of short fluorescent DNA probes is bound at the target RNA, robust signals are produced as opposed to the weak signals produced by a single probe
This feature enhances the signal-to-noise ratio to reveal the location of the target RNAs, even if a single probe may have off-target binding. smFISH provides information about the RNA abundance and subcellular localization of a given RNA at the single-molecule and single-cell levels
Sequential immunofluorescence (IF) and single-molecule RNA fluorescence in situ hybridization Note: We follow the sequential IF and smFISH protocol described by the manufacturer of smFISH probes (Biosearch Technologies, Petaluma, CA) with some modifications detailed below
Summary
4. Incubate cells (on a 12-mm circular coverslip) with 70 μl of primary antibody solution for 3 h at RT (e.g., 1:1,000 dilution of rabbit anti-PCNT antibody in 1x PBS in our example). 5. Perform three 5-min washes with 1x PBS and incubate the cells with 70 μl of secondary antibody solution overnight in the dark at 4 °C (e.g., 1:500 diluted anti-rabbit Alexa Fluor 488). To perform 5-min washes, gently transfer the coverslip to a well in a 24-well plate containing 1 ml of 1x PBS. Wash cells with Wash Buffer A for 5 min and incubate with 67 μl of Hybridization Buffer containing 125 nM smFISH DNA probe mix for 6 h at 37 °C in the dark. Note: For the best results, perform the incubations of antibody and smFISH probes in a humidified container (e.g., a 15-cm Petri dish with a piece of parafilm and wet paper towel inside, wrapped with aluminum foil to block light)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
