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Title: Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells

Abstract

Quantitative gene expression analysis in intact single cells can be achieved using single molecule- based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple FISH sub-probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific binding of sub-probes and tissue autofluorescence, limiting its accuracy. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on blinking frequencies of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses blinking frequency patterns, emitted from a transcript bound to multiple sub-probes, which are distinct from blinking patterns emitted from partial or nonspecifically bound sub-probes and autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2, and their ratio (Nkx2-2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct blinking frequency patterns, laying the foundation for reliable single-cellmore » transcriptomics.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1422272
Report Number(s):
PNNL-SA-126413
Journal ID: ISSN 0305-1048; KP1704020
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nucleic Acids Research; Journal Volume: 46; Journal Issue: 2
Country of Publication:
United States
Language:
English

Citation Formats

Cui, Yi, Hu, Dehong, Markillie, Lye Meng, Chrisler, William B., Gaffrey, Matthew J., Ansong, Charles, Sussel, Lori, and Orr, Galya. Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells. United States: N. p., 2017. Web. doi:10.1093/nar/gkx874.
Cui, Yi, Hu, Dehong, Markillie, Lye Meng, Chrisler, William B., Gaffrey, Matthew J., Ansong, Charles, Sussel, Lori, & Orr, Galya. Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells. United States. doi:10.1093/nar/gkx874.
Cui, Yi, Hu, Dehong, Markillie, Lye Meng, Chrisler, William B., Gaffrey, Matthew J., Ansong, Charles, Sussel, Lori, and Orr, Galya. Wed . "Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells". United States. doi:10.1093/nar/gkx874.
@article{osti_1422272,
title = {Fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for accurate detection and counting of RNA copies in single cells},
author = {Cui, Yi and Hu, Dehong and Markillie, Lye Meng and Chrisler, William B. and Gaffrey, Matthew J. and Ansong, Charles and Sussel, Lori and Orr, Galya},
abstractNote = {Quantitative gene expression analysis in intact single cells can be achieved using single molecule- based fluorescence in situ hybridization (smFISH). This approach relies on fluorescence intensity to distinguish between true signals, emitted from an RNA copy hybridized with multiple FISH sub-probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific binding of sub-probes and tissue autofluorescence, limiting its accuracy. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on blinking frequencies of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses blinking frequency patterns, emitted from a transcript bound to multiple sub-probes, which are distinct from blinking patterns emitted from partial or nonspecifically bound sub-probes and autofluorescence. Using multicolor fliFISH, we identified radial gene expression patterns in mouse pancreatic islets for insulin, the transcription factor, NKX2-2, and their ratio (Nkx2-2/Ins2). These radial patterns, showing higher values in β cells at the islet core and lower values in peripheral cells, were lost in diabetic mouse islets. In summary, fliFISH provides an accurate, quantitative approach for detecting and counting true RNA copies and rejecting false signals by their distinct blinking frequency patterns, laying the foundation for reliable single-cell transcriptomics.},
doi = {10.1093/nar/gkx874},
journal = {Nucleic Acids Research},
number = 2,
volume = 46,
place = {United States},
year = {Wed Oct 04 00:00:00 EDT 2017},
month = {Wed Oct 04 00:00:00 EDT 2017}
}