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

Journal Article · 04 October 2017 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkx874 · OSTI ID:1435646

Cui, Yi ^[1]; Hu, Dehong ^[1]; Markillie, Lye Meng ^[1]; Chrisler, William B. ^[1]; Gaffrey, Matthew J. ^[1]; Ansong, Charles ^[1]; Sussel, Lori ^[2]; Orr, Galya ^[1]

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1. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
2. Univ. of Colorado Denver, Aurora, CO (United States)

Here, 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 oligonucleotide probes, and background noise. Thus, the precision in smFISH is often compromised by partial or nonspecific probe binding and tissue autofluorescence, especially when only a small number of probes can be fitted to the target transcript. Here we provide an accurate approach for setting quantitative thresholds between true and false signals, which relies on on-off duty cycles of photoswitchable dyes. This fluctuation localization imaging-based FISH (fliFISH) uses on-time fractions (measured over a series of exposures) collected from transcripts bound to as low as 8 probes, which are distinct from on-time fractions collected from nonspecifically bound probes or 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 on-time fractions, laying the foundation for reliable single-cell transcriptomics.

• Research Organization: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
• Sponsoring Organization: USDOE
• Grant/Contract Number: AC05-76RL01830
• OSTI ID: 1435646
• Report Number(s): PNNL-SA-126413
• Journal Information: Nucleic Acids Research, Vol. 46, Issue 2; ISSN 0305-1048
• Publisher: Oxford University PressCopyright Statement
• Country of Publication: United States
• Language: English