Simultaneous Detection of mRNA and Protein in S. cerevisiae by Single-Molecule FISH and Immunofluorescence

Evelina Tutucci, Robert H. Singer

Research output: Chapter in Book / Report / Conference proceedingChapterAcademicpeer-review

Abstract

Single-molecule fluorescent in situ hybridization (smFISH) enables the detection and quantification of endogenous mRNAs within intact fixed cells. This method utilizes tens of singly labeled fluorescent DNA probes hybridized against the mRNA of interest, which can be detected by using standard wide-field fluorescence microscopy. This approach provides the means to generate absolute quantifications of gene expression within single cells, which can be used to link molecular fluctuations to phenotypes. To be able to correlate the expression of an mRNA and a protein of interest in individual cells, we combined smFISH with immunofluorescence (IF) in yeast cells. Here, we present our smFISH-IF protocol to visualize and quantify two cell cycle-controlled mRNAs (CLN2 and ASH1) and the cell cycle marker alpha-tubulin in S. cerevisiae. This protocol, which is performed over 2 days, can be used to visualize up to three colors at the time (i.e., two mRNAs, one protein). Even if the described protocol is designed for S. cerevisiae, we think that the considerations discussed here can be useful to develop and troubleshoot smFISH-IF protocols for other model organisms.

Original languageEnglish
Title of host publicationRNA Tagging
Subtitle of host publicationMethods and Protocols
EditorsManfred Heinlein
Place of PublicationNew York, NY
PublisherHumana
Pages51-69
Number of pages19
ISBN (Electronic)9781071607121
ISBN (Print)9781071607114
DOIs
Publication statusPublished - 2020

Publication series

NameMethods in molecular biology (MIMB)
Volume2166
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Cell cycle
  • Immunofluorescence
  • RNA FISH
  • RNA localization
  • S. cerevisiae
  • Single molecule
  • Single-cell imaging
  • smFISH
  • smFISH-IF

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