smiFISH and FISH-quant – a flexible single RNA detection approach with super-resolution capability

Tsanov, Nikolay; Samacoïts, Aubin; Chouaib, Racha; Traboulsi, Abdel-Meneem; Gostan, Thierry; Weber, Christian; Zimmer, Christophe; Zibara, Kazem; Walter, Thomas; Peter, Matthias; Bertrand, Édouard; Mueller, Florian

doi:10.1093/nar/gkw784

Cited by 360 publications

(492 citation statements)

References 27 publications

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“…For this reason, a clearing method that preserves RNAs while removing proteins and lipids is desired for RNA FISH imaging. In the recently developed expansion microscopy method, proteins (25) and, more recently, RNAs (26,27) are physically anchored to a solvent-expandable and clearable poly-electrolyte matrix, effectively imprinting signals of these components on this matrix and allowing these molecular signals to be expanded along with the matrix for increasing image resolution. Inspired by this approach, we anchored RNA molecules to a nonswellable polyacrylamide (PA) matrix and then removed unwanted, non-RNA components, such as proteins and lipids, with the aim to remove their contribution to background fluorescence.…”

Section: Significancementioning

confidence: 99%

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

Moffitt

Hao

Bambah-Mukku

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

242

249

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Highly multiplexed single-molecule FISH has emerged as a promising approach to spatially resolved single-cell transcriptomics because of its ability to directly image and profile numerous RNA species in their native cellular context. However, backgroundfrom off-target binding of FISH probes and cellular autofluorescence-can become limiting in a number of important applications, such as increasing the degree of multiplexing, imaging shorter RNAs, and imaging tissue samples. Here, we developed a sample clearing approach for FISH measurements. We identified off-target binding of FISH probes to cellular components other than RNA, such as proteins, as a major source of background. To remove this source of background, we embedded samples in polyacrylamide, anchored RNAs to this polyacrylamide matrix, and cleared cellular proteins and lipids, which are also sources of autofluorescence. To demonstrate the efficacy of this approach, we measured the copy number of 130 RNA species in cleared samples using multiplexed error-robust FISH (MERFISH). We observed a reduction both in the background because of off-target probe binding and in the cellular autofluorescence without detectable loss in RNA. This process led to an improved detection efficiency and detection limit of MERFISH, and an increased measurement throughput via extension of MERFISH into four color channels. We further demonstrated MERFISH measurements of complex tissue samples from the mouse brain using this matrix-imprinting and -clearing approach. We envision that this method will improve the performance of a wide range of in situ hybridization-based techniques in both cell culture and tissues.tissue clearing | fluorescence in situ hybridization | multiplexed imaging | single-cell transcriptomics | brain S ingle-molecule FISH (smFISH) is a powerful technique that allows the direct imaging of individual RNA molecules within single cells (1, 2). In this approach, RNAs are labeled via the hybridization of fluorescently labeled oligonucleotide probes, producing bright fluorescent spots for single RNA molecules, which reveal both the abundance and the spatial distribution of these RNAs inside cells (1, 2). The ability of smFISH to image gene expression at the single-cell level in both cell culture and tissue has led to exciting advances in our understanding of the natural noise in gene expression and its role in cellular response (3, 4), the intracellular spatial organization of RNAs and its role in posttranscriptional regulation (5, 6), and the spatial variation in gene expression within complex tissues and its role in the molecular definition of cell types and tissue functions (6, 7).To extend the benefits of this technique to systems-level questions and high-throughput gene-expression profiling, approaches to increase the multiplexing of smFISH (i.e., the number of different RNA species that can be simultaneously quantified within the same cell) have been developed (8-13). Most of these approaches take advantage of color multiplexing, which has allowed a few tens...

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Section: Significancementioning

confidence: 99%

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

Moffitt

Hao

Bambah-Mukku

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

242

249

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“…Recently, other cost-reducing RNA detection techniques with single-molecule sensitivity have been developed, such as the single-molecule inexpensive FISH (smiFISH) (Tsanov et al 2016) and single-molecule hybridization chain reaction (smHCR) (Shah et al 2016). These two techniques also use inexpensive unlabeled oligonucleotides to initiate the RNA detection.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, smiFISH produces a signal roughly twice as bright as smFISH carried out by the same number of singly labeled probes, with an identical signal-to-noise ratio. smiFISH also preserves the quantitative feature of smFISH, which directly relates signal intensity to RNA copy number (Tsanov et al 2016). smHCR, on the other hand, has greatly increased brightness due to the series of fluorophores deployed around the target during the chain reaction.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic production of single-molecule FISH and RNA capture probes

Gáspár

Wippich

Ephrussi

2017

RNA

144

100

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Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.

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“…Currently, the smFISH probe selection is based on melting temperature (Tm) 3 or Gibbs free energy 10 , which are not very indicative of probe hybridization efficiency. We developed a pipeline based on Primer3 11 and DECIPHER 12 to design and filter for GSO with high hybridization efficiency, which is a more tangible indicator (Figure 1c).…”

Section: Cc-by-nc-ndmentioning

confidence: 99%

Autonomous combinatorial color barcoding for multiplexing single molecule RNA visualization

Cheng

Zhuo

Hartmann

et al. 2017

Preprint

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Single molecular fluorescence in situ hybridization (smFISH) detects RNA transcripts with spatial information and digital molecular counting. However, the broad usage of smFISH is still hindered by the complex chemical probe conjugation or microscopy set-up, especially for investigating multiple gene expression. Here we present a multiple fluorophore enzymatic labeling method (termed HuluFISH) for smFISH probes to achieve flexible combinatorial color barcoding in single hybridization step. The multiplex capacity of HuluFISH follows an exponential growth with the increase of the number of fluorophore types. We demonstrate that this method can be used to detect cellular heterogeneity in embryonic mouse brain on single cell level.

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smiFISH and FISH-quant – a flexible single RNA detection approach with super-resolution capability

Cited by 360 publications

References 27 publications

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing

Enzymatic production of single-molecule FISH and RNA capture probes

Autonomous combinatorial color barcoding for multiplexing single molecule RNA visualization

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