title: Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription.
abstract: The steroidogenic acute regulatory protein (StAR) has been proposed to serve as the switch that can turn on/off steroidogenesis. We investigated the events that facilitate dynamic StAR transcription in response to cAMP stimulation in MA-10 Leydig cells, focusing on splicing anomalies at StAR gene loci. We used 3' reverse primers in a single reaction to respectively quantify StAR primary (p-RNA), spliced (sp-RNA/mRNA), and extended 3' untranslated region (UTR) transcripts, which were quantitatively imaged by high-resolution fluorescence in situ hybridization (FISH). This approach delivers spatio-temporal resolution of initiation and splicing at single StAR loci, and transfers individual mRNA molecules to cytoplasmic sites. Gene expression was biphasic, initially showing slow splicing, transitioning to concerted splicing. The alternative 3.5-kb mRNAs were distinguished through the use of extended 3'UTR probes, which exhibited distinctive mitochondrial distribution. Combining quantitative PCR and FISH enables imaging of localization of RNA expression and analysis of RNA processing rates.
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53706, United States; Endocrinology and Reproductive Physiology Program, University of Wisconsin, Madison, WI 53706, United States. Electronic address: jwlee7@wisc.edu.
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53706, United States.
authors.forename: Yee Hoon
authors.initials: YH
authors.lastname: Foong
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53706, United States.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Musaitif
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53706, United States.
authors.forename: Tiegang
authors.initials: T
authors.lastname: Tong
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI 53706, United States; Endocrinology and Reproductive Physiology Program, University of Wisconsin, Madison, WI 53706, United States; Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706, United States.
authors.forename: Colin
authors.initials: C
authors.lastname: Jefcoate
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D020029:Base Pairing / Q000235:genetics; D002478:Cells, Cultured; D021621:Imaging, Three-Dimensional; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D008853:Microscopy / Q000379:methods*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics*; D026723:Polyadenylation / Q000187:drug effects / Q000235:genetics; D012313:RNA / Q000378:metabolism*; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects; D000098682:Steroidogenic Acute Regulatory Protein
chemical_list: D020413:3' Untranslated Regions; D010750:Phosphoproteins; D012333:RNA, Messenger; D000098682:Steroidogenic Acute Regulatory Protein; D015124:8-Bromo Cyclic Adenosine Monophosphate; D012313:RNA
keywords: Fluorescence in situ hybridization; PCR; Splicing; StAR; Transcription