Imaging individual mRNA molecules using multiple singly labeled probes

By Arjun Raj, P. V. D. van den Bogaard, Scott A. Rifkin, Alexander van Oudenaarden, Sanjay Tyagi. Nature Methods, 2008

1,831 citations recorded by the source corpus.

We describe a method for imaging individual mRNA molecules in fixed cells by probing each mRNA species with 48 or more short, singly labeled oligonucleotide probes. This makes each mRNA molecule visible as a computationally identifiable fluorescent spot by fluorescence microscopy. We demonstrate simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, and mammalian cell lines and neurons.

Subfields: Biophysics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/7500df32949d73b880bd6da0dbf3952f2b12a0d6

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Citations in the corpus

The Influenza A Virus PB2, PA, NP, and M Segments Play a Pivotal Role during Genome Packaging (2012, methodology): “Since both RNA gel silver staining and qRT-PCR techniques measured the efficiency of vRNA incorporation in a virus population, we conducted smFISH experiments (2, 31) to analyze the incorporated viral RNAs in individual virus particles and measured the efficiencies of two viral RNAs being copackaged”
Brief update on endocytosis of nanomedicines (2019, background): “In fixed cells, single molecule FISH (smFISH) makes individual mRNA transcripts appear as visible fluorescent spots that can be readily analyzed by fluorescence microscopy [262].”
Genomic maps of lincRNA occupancy reveal principles of RNA-chromatin interactions (2011, methodology): “We were inspired by the technique of single-molecule RNA fluorescent in situ hybridization (Fusco et al., 2004; Raj et al., 2008), where dozens of short oligonucleotide probes that tile the length of an RNA generate highly specific signals.”
Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. (2012, methodology): “FISH Imaging and Analysis We performed FISH imaging and analysis as described previously (Raj et al., 2008, 2010) and in the Extended Experimental Procedures.”
Spatial transcriptome profiling by MERFISH reveals subcellular RNA compartmentalization and cell cycle-dependent gene expression (2019, background): “In each round, RNAs are imaged by FISH at the single-molecule level (13, 14), and the use of error-robust barcodes allows errors accumulated through multiple imaging rounds to be detected and/or corrected (6).”
Programmable in situ amplification for multiplexed imaging of mRNA expression (2010, positive): “The observation that autofluorescence is the dominant source of background suggests that addressing each target mRNA with a probe set comprising multiple probes 13,19 would further increase the signal-to-background ratio.”
Single-Cell and Single-Molecule Analysis of Gene Expression Regulation (2016, methodology): “Single-mRNA molecules are detected in fixed cells by smFISH (50, 121) (Figure 1a).”
Genome-wide RNA Tomography in the zebrafish embryo. (2014, methodology): “Tissues composed of different cell types may also benefit from methods with higher spatial resolution such as single-molecule RNA FISH (Lyubimova et al., 2013; Raj et al., 2008).”
Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells (2015, methodology): “Single-cell measurements of gene expression, using imaging techniques such as RNA-FiSH (fluorescence in situ hybridization), have provided important insights into the kinetics of transcription and cell-to-cell variation in gene expression 1–3 .”
Large‐scale image‐based screening and profiling of cellular phenotypes (2017, background): “The signal intensity of FISH can be increased by using multiple short oligonucleotide probes to adjacent sequences on a RNA target (13), and/or conjugating multiple fluorescent dyes to each oligonucleotide probe (14).”
RNA Phase Transitions in Repeat Expansion Disorders (2017, positive): “For RNA FISH in U-2OS cells, cells expressing the desired RNA (induced for 24 hours) were fixed with 2% paraformaldehyde for 10 min at room temperature and permeabilized by overnight incubation in 70% ethanol at 4°C. Alternatively, cells were fixed and permeabilized by incubation for 10 min in metha”
The technology and biology of single-cell RNA sequencing. (2015, methodology): “PCR, used in SmartSeq, its optimized version SmartSeq2 (Picelli et al., 2013; Ramsköld et al., 2012), STRT (Islam et al., 2012), the Tang protocol (Tang et al., 2009), and SC3-seq (Nakamura et al., 2015), is a nonlinear amplification process, and its efficiency is sequence dependent.”
Design and Analysis of Single-Cell Sequencing Experiments. (2015, methodology): “Single-cell analysis of a small number of genes can be performed with imaging-based methods such as single-molecule ﬂuorescence in situ hybridization (Raj et al., 2008) or by ﬂow cytometry, exploiting cell surface markers or ﬂuorescent reporter proteins.”