ClampFISH detects individual nucleic acid molecules using click chemistry–based amplification

Rouhanifard, Sara H.; Mellis, Ian A.; Dunagin, Margaret C.; Bayatpour, Sareh; Jiang, Connie; Dardani, Ian; Symmons, Orsolya; Emert, Benjamin; Torre, Enrique; Coté, Allison; Sullivan, Alessandra M.; Stamatoyannopoulos, J; Raj, Arjun

doi:10.1038/nbt.4286

Cited by 117 publications

(114 citation statements)

References 28 publications

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“…This protocol for generating barcode clampFISH probes is also available online at https://www.protocols.io/view/invertedclampfish-ligation-qxwdxpe . We prepared amplifier probes MM2B, MM2C, P9B and P9C as described previously (Rouhanifard et al, 2018) .…”

Section: Barcode Fish Probe Designmentioning

confidence: 99%

Variability within rare cell states enables multiple paths towards drug resistance

Emert

Coté

Torre

et al. 2020

Preprint

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Molecular differences between individual cells can lead to dramatic differences in cell fate following an applied treatment, such as the difference between death versus survival of cancer cells upon receiving anti-cancer drugs. However, current strategies to retrospectively identify the cells that give rise to distinct rare behaviors and determine their distinguishing molecular characteristics remain limited. Here we describe Rewind, a methodology that combines genetic barcoding with an RNA-based readout to directly capture rare cells that give rise to cellular behaviors of interest, specifically the emergence of resistance to targeted cancer therapy. Using Rewind, we analyzed over 5 million cells to identify differences in gene expression and MAP-kinase signaling in single melanoma cells that mark a rare subpopulation of drug-naive cells (initial frequency of ~1:1000-1:10,000 cells) that ultimately gives rise to drug resistant clones. We further show that even within this rare subpopulation, molecular differences between single cells before the application of drug predict future differences in drug resistant behavior. Similarly, we show that treatments that modify the frequency of resistance can allow otherwise non-resistant cells in the drug-naive population to become resistant, and that these new populations are marked by the variable expression of distinct genes. Together, our results reveal the presence of cryptic variability that can underlie a range of distinct rare-cell phenotypic outcomes upon drug exposure. Applying Rewind to other rare biological phenomena, such as cancer metastasis, tissue regeneration, and stem cell reprogramming, may provide a means to map rare cellular states to the unique cellular fates to which they give rise.

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Section: Barcode Fish Probe Designmentioning

confidence: 99%

Variability within rare cell states enables multiple paths towards drug resistance

Emert

Coté

Torre

et al. 2020

Preprint

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“…To increase signal strength in FISH, a number of signal amplification schemes that restrict the amplified signal to the site of the target have been developed. They include the hybridization chain reaction (HCR), branched DNA amplification, clamp-FISH, and rolling circle amplification (8)(9)(10)(11)(12)(13). However, nonspecifically bound probes can also trigger amplification, giving rise to false signals that increase as the number of probes is increased (14).…”

mentioning

confidence: 99%

High-fidelity amplified FISH for the detection and allelic discrimination of single mRNA molecules

Marras¹,

Bushkin²,

Tyagi³

2019

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

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Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with an HCR initiator sequence are prone to producing false signals. Here we describe a system of interacting hairpin binary probes in which the HCR initiator sequence is conditionally sequestered. The binding of these probes to a perfectly complementary target unmasks the initiator, enabling the generation of an amplified signal. This probe system can distinguish single-nucleotide variations within single mRNA molecules and produces amplified signals in situ for both mutant and wild-type variants, each in a distinguishable color. This technology will augment studies of imbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By tiling these probes along the length of an mRNA target, enhanced signals can be obtained, thereby enabling the scanning of tissue sections for gene expression utilizing lower magnification microscopy, overcoming tissue autofluorescence, and allowing the detection of low-abundance biomarkers in flow cytometry.

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“…Since commercial ligases such as T4 DNA ligase, T4 RNA ligase 2 and Ampligase show low activity on DNA/RNA hybrids, RNA has to be reverse-transcribed to cDNA fragments before introducing padlock probes (3). However, cDNA synthesis on fixed tissue sections is a challenging and expensive procedure (26), prompting new elegant approaches trying to circumvent reverse-transcription by introducing Click-chemistry to ligate DNA probes after hybridization on their RNA targets (ClampFISH (27)). More recently, “H-type” DNA probes, which are hybridized to both RNA and padlock probes (PLISH (28)), or SNAIL-design probes have been successfully used to facilitate intramolecular ligation of the padlock probes (STARmap (25)).…”

Section: Introductionmentioning

confidence: 99%

SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

Sountoulidis¹,

Liontos²,

Nguyen³

et al. 2020

Preprint

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16 17 ǂ Corresponding authors: Christos Samakovlis (christos.samakovlis@scilifelab.se), Alexandros 18 Sountoulidis (alexandros.sountoulidis@scilifelab.se) 19 20 Keywords: SCRINSHOT, padlock-probe, multiplex RNA FISH, spatial cell-type map, lung 21 22 2 23 Abstract 24 Changes in cell identities and positions underlie tissue development and disease progression. 25 Although, single-cell mRNA sequencing (scRNA-Seq) methods rapidly generate extensive lists of 26 cell-states, spatially resolved single-cell mapping presents a challenging task. We developed 27 SCRINSHOT (Single Cell Resolution IN Situ Hybridization On Tissues), a sensitive, multiplex 28 RNA mapping approach. Direct hybridization of padlock probes on mRNA is followed by 29 circularization with SplintR ligase and rolling circle amplification (RCA) of the hybridized padlock 30 probes. Sequential detection of RCA-products using fluorophore-labeled oligonucleotides profiles 31 thousands of cells in tissue sections. We evaluated SCRINSHOT specificity and sensitivity on 32 murine and human organs. SCRINSHOT quantification of marker gene expression shows high 33 correlation with published scRNA-Seq data over a broad range of gene expression levels. We 34 demonstrate the utility of SCRISHOT by mapping the locations of abundant and rare cell types 35 along the murine airways. The amenability, multiplexity and quantitative qualities of SCRINSHOT 36 facilitate single cell mRNA profiling of cell-state alterations in tissues under a variety of native and 37 experimental conditions. 38 39 40 41 42 43 44 45 65addressed by the sequential fluorescence in situ hybridization (seqFISH) (14, 15) and the 66 multiplexed error-robust FISH (MERFISH) (4). These approaches utilize sequential rounds of 67 hybridization of FISH probes or barcode-based primary probes to detect multiple RNA species. 68The outstanding throughput of these methods makes them strong candidates for generation of 69 spatial transcriptome maps in tissues. However, since the principle of these techniques is similar 70 to smFISH, they require large number of gene-specific probes, confocal or super-resolution 4 71 microscopy to deconvolve the signals and complicated algorithms for both probe design and 72 analysis. Nevertheless, the low signal-to-noise ratio still remains a major technical challenge of 73 these methods, especially for tissue sections with strong auto-fluorescence from structural 74 extracellular matrix components like collagen and elastin (16). New strategies for signal 75 amplifications, such as branched-DNA amplification (RNAScope) (17) and hybridization chain 76 reaction (18, 19), have been recently combined with sophisticated probe design (AmpFISH) (20) 77 to increase sensitivity and specificity of smFISH. 78 Padlock probes have been successfully used to detect RNA species (21). They are linear DNA 79 molecules, with complementary arms to the target mRNA sequence and a common "backbone". 80 Upon hybridization with the target sequence, they can be ligated, creating circular single-stranded 81...

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ClampFISH detects individual nucleic acid molecules using click chemistry–based amplification

Cited by 117 publications

References 28 publications

Variability within rare cell states enables multiple paths towards drug resistance

Variability within rare cell states enables multiple paths towards drug resistance

High-fidelity amplified FISH for the detection and allelic discrimination of single mRNA molecules

SCRINSHOT, a spatial method for single-cell resolution mapping of cell states in tissue sections

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