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Updated: Sep 10, 2026
Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH
Published on: September 20, 2020
Protocol for measuring endocrine-disruptive effects on transcriptional bursting using single-molecule imaging in
Pelin Yaşar1, Christopher R Day1, Joseph Rodriguez1
1Single Cell Dynamics Group, Epigenetics and RNA Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, NC 27709, USA.
Abstract:
Transcriptional bursts regulate gene expression by altering burst size or burst frequency. Here, we present a protocol to analyze estrogen-responsive transcriptional bursting of the TFF1 gene in human breast cancer cell lines by integrating fixed-cell single-molecule RNA fluorescence in situ hybridization (smFISH) and live-cell single-molecule imaging. We describe cell seeding, ligand dose-response treatment, and smFISH hybridization and imaging to quantify transcription sites. We then detail live-cell imaging of a TFF1-MS2 reporter and integration of both datasets to resolve how endocrine disruptors modulate bursting dynamics. For complete details on the use and execution of this protocol, please refer to Day et al.1.
