Main Menu (Mobile)- Block
- Our Research
-
Support Teams
- Overview
- Anatomy and Histology
- Cryo-Electron Microscopy
- Electron Microscopy
- Flow Cytometry
- Fly Facility
- Gene Targeting and Transgenics
- Immortalized Cell Line Culture
- Integrative Imaging
- Janelia Experimental Technology
- Media Prep
- Molecular Genomics
- Primary & iPS Cell Culture
- Project Pipeline Support
- Project Technical Resources
- Quantitative Genomics
- Scientific Computing Software
- Scientific Computing Systems
- Viral Tools
- Vivarium
- Open Science
- You + Janelia
- About Us
Main Menu - Block
- Overview
- Anatomy and Histology
- Cryo-Electron Microscopy
- Electron Microscopy
- Flow Cytometry
- Fly Facility
- Gene Targeting and Transgenics
- Immortalized Cell Line Culture
- Integrative Imaging
- Janelia Experimental Technology
- Media Prep
- Molecular Genomics
- Primary & iPS Cell Culture
- Project Pipeline Support
- Project Technical Resources
- Quantitative Genomics
- Scientific Computing Software
- Scientific Computing Systems
- Viral Tools
- Vivarium
Note: Research in this publication was not performed at Janelia.
Abstract
Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d.
