title: Single molecule approaches for quantifying transcription and degradation rates in intact mammalian tissues.
abstract: A key challenge in mammalian biology is to understand how rates of transcription and mRNA degradation jointly shape cellular gene expression. Powerful techniques have been developed for measuring these rates either genome-wide or at the single-molecule level, however these techniques are not applicable to assessment of cells within their native tissue microenvironment. Here we describe a technique based on single molecule Fluorescence in-situ Hybridization (smFISH) to measure transcription and degradation rates in intact mammalian tissues. The technique is based on dual-color libraries targeting the introns and exons of the genes of interest, enabling visualization and quantification of both nascent and mature mRNA. We present a software, TransQuant, that facilitates quantifying these rates from smFISH images. Our approach enables assessment of both transcription and degradation rates of any gene of interest while controlling for the inherent heterogeneity of intact tissues.
authors.affiliation: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Keren
authors.initials: K
authors.lastname: Bahar Halpern
authors.affiliation: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel. Electronic address: shalev.itzkovitz@weizmann.ac.il.
authors.forename: Shalev
authors.initials: S
authors.lastname: Itzkovitz
mesh_terms: D001275:ATP Citrate (pro-S)-Lyase / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001124:Argininosuccinate Synthase / Q000235:genetics* / Q000378:metabolism; D060833:Cellular Microenvironment; D005091:Exons; D005456:Fluorescent Dyes / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods* / Q000706:statistics & numerical data; D007438:Introns; D008099:Liver / Q000378:metabolism; D051379:Mice; D015335:Molecular Probes / Q000737:chemistry; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods* / Q000706:statistics & numerical data; D054852:Small Molecule Libraries / Q000737:chemistry; D012984:Software; D049490:Systems Biology / Q000379:methods; D014158:Transcription, Genetic
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes; D012333:RNA, Messenger; D054852:Small Molecule Libraries; D001275:ATP Citrate (pro-S)-Lyase; D001124:Argininosuccinate Synthase
keywords: Single molecule; Systems biology; Transcription