Single-mRNA counting using fluorescent in situ hybridization in budding yeast.
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.
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA.
Tatjana
T
Trcek
Jeffrey A
JA
Chao
Daniel R
DR
Larson
Hye Yoon
HY
Park
Daniel
D
Zenklusen
Shailesh M
SM
Shenoy
Robert H
RH
Singer
D000465:Algorithms; D016375:Antisense Elements (Genetics); D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D012331:RNA, Fungal / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D016375:Antisense Elements (Genetics); D012331:RNA, Fungal; D012333:RNA, Messenger