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Qian Zhu
Cells in complex tissues are organized by distinct microenvironments and anatomical structures. This spatial environment of cells is thought to be important for division of labor and other specialized functions of tissues. Recently developed spatial transcriptomic technologies enable the quantification of expression of hundreds of genes while accounting for cells' spatial coordinates, providing an opportunity to study spatially organized structures. Here, we describe a computational pipeline for detecting the spatial organization of cells based on a hidden Markov random field model...
2019: Methods in Molecular Biology
Laura I Hudish, David S Lorberbaum, Lori Sussel
β cell heterogeneity has emerged as an important contributor to islet function, with potential implications for diabetes. Using an optimized smFISH technique in intact islets, Farack et al. (2018) identify in the islet core an endocrine cell population of "extreme" β cells with distinct molecular properties.
January 7, 2019: Developmental Cell
Masahiro Satoh, Seitaro Nomura, Mutsuo Harada, Toshihiro Yamaguchi, Toshiyuki Ko, Tomokazu Sumida, Haruhiro Toko, Atsuhiko T Naito, Norifumi Takeda, Takashige Tobita, Takanori Fujita, Masamichi Ito, Kanna Fujita, Masato Ishizuka, Taro Kariya, Hiroshi Akazawa, Yoshio Kobayashi, Hiroyuki Morita, Eiki Takimoto, Hiroyuki Aburatani, Issei Komuro
BACKGROUND: The heart responds to hemodynamic overload through cardiac hypertrophy and activation of the fetal gene program. However, these changes have not been thoroughly examined in individual cardiomyocytes, and the relation between cardiomyocyte size and fetal gene expression remains elusive. We established a method of high-throughput single-molecule RNA imaging analysis of in vivo cardiomyocytes and determined spatial and temporal changes during the development of heart failure...
January 3, 2019: Journal of Molecular and Cellular Cardiology
Chenglin Wu, Michele Simonetti, Carla Rossell, Marco Mignardi, Reza Mirzazadeh, Laura Annaratone, Caterina Marchiò, Anna Sapino, Magda Bienko, Nicola Crosetto, Mats Nilsson
Single-molecule RNA fluorescence in situ hybridization (smFISH) represents a promising approach to quantify the expression of clinically useful biomarkers in tumor samples. However, routine application of smFISH to formalin-fixed, paraffin-embedded (FFPE) samples is challenging due to the low signal intensity and high background noise. Here we present RollFISH, a method combining the specificity of smFISH with the signal boosting of rolling circle amplification. We apply RollFISH to quantify widely used breast cancer biomarkers in cell lines and FFPE samples...
2018: Communications biology
Aubin Samacoits, Racha Chouaib, Adham Safieddine, Abdel-Meneem Traboulsi, Wei Ouyang, Christophe Zimmer, Marion Peter, Edouard Bertrand, Thomas Walter, Florian Mueller
RNA localization is a crucial process for cellular function and can be quantitatively studied by single molecule FISH (smFISH). Here, we present an integrated analysis framework to analyze sub-cellular RNA localization. Using simulated images, we design and validate a set of features describing different RNA localization patterns including polarized distribution, accumulation in cell extensions or foci, at the cell membrane or nuclear envelope. These features are largely invariant to RNA levels, work in multiple cell lines, and can measure localization strength in perturbation experiments...
November 2, 2018: Nature Communications
Gunjan D Mehta, David A Ball, Peter R Eriksson, Razvan V Chereji, David J Clark, James G McNally, Tatiana S Karpova
It is unknown how the dynamic binding of transcription factors (TFs) is molecularly linked to chromatin remodeling and transcription. Using single-molecule tracking (SMT), we show that the chromatin remodeler RSC speeds up the search process of the TF Ace1p for its response elements (REs) at the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates transcription bursts of CUP1 only by modulating TF occupancy but does not affect initiation and elongation rates...
September 27, 2018: Molecular Cell
Evelina Tutucci, Maria Vera, Robert H Singer
The MS2 system has been widely used, in organisms ranging from bacteria to higher eukaryotes, to image single mRNAs in intact cells with high precision. We have recently re-engineered the MS2 system for accurate detection of mRNAs in living Saccharomyces cerevisiae. Previous MS2 systems affected the degradation of the tagged mRNA, which led to accumulation of MS2 fragments and to erroneous conclusions about mRNA localization and expression. Here we describe a step-by-step protocol for the use of our latest MS2 system (MBSV6) for detecting endogenously tagged mRNAs using wide-field fluorescent microscopy in living yeast...
October 2018: Nature Protocols
Yi-Ying Chou, Timothée Lionnet
Influenza A virus is an enveloped virus with a segmented genome consisting of eight negative-sense, single-stranded RNAs. Accumulating evidence has revealed that influenza viruses selectively package their genomes. However, less is known about how different viral RNA segments are selected for incorporation into progeny virions. Understanding the trafficking routes and assembly process of various viral RNA segments during infection will shed light on the mechanisms of selective genome packaging for influenza A viruses...
2018: Methods in Molecular Biology
Nikhita Pasnuri, Purnati Khuntia, Aprotim Mazumder
Single molecule Fluorescence in situ Hybridization (smFISH) for mRNA provides a powerful quantitative handle on expression from endogenous gene loci. While the method has been widely applied in cells in culture, applications to primary tissue samples remain fewer, and often use involved cryosectioning. Even apart from quantitative access to absolute transcript counts in specific tissue volumes, many other advantages of smFISH can be envisaged in tissue samples. Primary among these are the ability to report on subtle differences in expression among different cell types within a tissue, and the ability to correlate the expression from different target genes...
October 2018: Mechanisms of Development
Xavier Pichon, Mounia Lagha, Florian Mueller, Edouard Bertrand
The spatiotemporal regulation of gene expression is key to many biological processes. Recent imaging approaches opened exciting perspectives for understanding the intricate mechanisms regulating RNA metabolism, from synthesis to decay. Imaging techniques allow their observation at high spatial and temporal resolution, while keeping cellular morphology and micro-environment intact. Here, we focus on approaches for imaging single RNA molecules in cells, tissues, and embryos. In fixed cells, the rapid development of smFISH multiplexing opens the way to large-scale single-molecule studies, while in live cells, gene expression can be observed in real time in its native context...
August 2, 2018: Molecular Cell
Samuel E Taylor, Ruth D Taylor, Jack Price, Laura C Andreae
BACKGROUND: Deletions and mutations in the SHANK3 gene are strongly associated with autism spectrum disorder and underlie the autism-associated disorder Phelan-McDermid syndrome. SHANK3 is a scaffolding protein found at the post-synaptic membrane of excitatory neurons. METHODS: Single-molecule fluorescence in-situ hybridization (smFISH) allows the visualization of single mRNA transcripts in vitro. Here we perform and quantify smFISH in human inducible pluripotent stem cell (hiPSC)-derived cortical neurons, targeting the SHANK3 transcript...
July 31, 2018: Stem Cell Research & Therapy
Jingxun Chen, David McSwiggen, Elçin Ünal
Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene NDC80 during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform...
May 25, 2018: Journal of Visualized Experiments: JoVE
Aouefa Amoussouvi, Lotte Teufel, Matthias Reis, Martin Seeger, Julia Katharina Schlichting, Gabriele Schreiber, Andreas Herrmann, Edda Klipp
Gene expression is a stochastic process and its appropriate regulation is critical for cell cycle progression. Cellular stress response necessitates expression reprogramming and cell cycle arrest. While previous studies are mostly based on bulk experiments influenced by synchronization effects or lack temporal distribution, time-resolved methods on single cells are needed to understand eukaryotic cell cycle in context of noisy gene expression and external perturbations. Using smFISH, microscopy and morphological markers, we monitored mRNA abundances over cell cycle phases and calculated transcriptional noise for SIC1 , CLN2 , and CLB5 , the main G1/S transition regulators in budding yeast...
2018: NPJ Systems Biology and Applications
Christoph G Engert, Rita Droste, Alexander van Oudenaarden, H Robert Horvitz
To better understand the tissue-specific regulation of chromatin state in cell-fate determination and animal development, we defined the tissue-specific expression of all 36 C. elegans presumptive lysine methyltransferase (KMT) genes using single-molecule fluorescence in situ hybridization (smFISH). Most KMTs were expressed in only one or two tissues. The germline was the tissue with the broadest KMT expression. We found that the germline-expressed C. elegans protein SET-17, which has a SET domain similar to that of the PRDM9 and PRDM7 SET-domain proteins, promotes fertility by regulating gene expression in primary spermatocytes...
April 2018: PLoS Genetics
Benjamin R King, Aubin Samacoits, Philip L Eisenhauer, Christopher M Ziegler, Emily A Bruce, Daniel Zenklusen, Christophe Zimmer, Florian Mueller, Jason Botten
Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were based largely on viral RNA profiling data derived from entire populations of cells...
June 15, 2018: Journal of Virology
Guiping Wang, Jeffrey R Moffitt, Xiaowei Zhuang
As an image-based single-cell transcriptomics approach, multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows hundreds to thousands of RNA species to be identified, counted and localized in individual cells while preserving the native spatial context of RNAs. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule FISH (smFISH) to read out these barcodes. The accuracy of RNA identification relies on spatially separated signals from individual RNA molecules, which limits the density of RNAs that can be measured and makes the multiplexed imaging of a large number of high-abundance RNAs challenging...
March 19, 2018: Scientific Reports
Seongjin Park, Magda Bujnowska, Eric L McLean, Jingyi Fei
We present a method for the quantification of small regulatory RNAs (sRNAs) in bacteria, by combining single-molecule fluorescence in situ hybridization (smFISH), super-resolved single-fluorophore microscopy, and clustering analysis. Compared to smFISH imaging with diffraction-limited fluorescence microscopy, our method provides better quantification for short and abundant RNA (such as sRNAs) in a small volume of bacterial cells. Our method can also be directly used for the quantification of messenger RNAs (mRNAs)...
2018: Methods in Molecular Biology
Kyung Won Kim, Ngang Heok Tang, Matthew G Andrusiak, Zilu Wu, Andrew D Chisholm, Yishi Jin
The PIWI-interacting RNA (piRNA) pathway has long been thought to function solely in the germline, but evidence for its functions in somatic cells is emerging. Here we report an unexpected role for the piRNA pathway in Caenorhabditis elegans sensory axon regeneration after injury. Loss of function in a subset of components of the piRNA pathway results in enhanced axon regrowth. Two essential piRNA factors, PRDE-1 and PRG-1/PIWI, inhibit axon regeneration in a gonad-independent and cell-autonomous manner. By smFISH analysis we find that prde-1 transcripts are present in neurons, as well as germ cells...
February 7, 2018: Neuron
Rinat Arbel-Goren, Yonatan Shapira, Joel Stavans
A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes...
December 21, 2017: Journal of Visualized Experiments: JoVE
Tatjana Trcek, Samir Rahman, Daniel Zenklusen
Cellular mRNA levels are determined by the rates of mRNA synthesis and mRNA decay. Typically, mRNA degradation kinetics are measured on a population of cells that are either chemically treated or genetically engineered to inhibit transcription. However, these manipulations can affect the mRNA decay process itself by inhibiting regulatory mechanisms that govern mRNA degradation, especially if they occur on short time-scales. Recently, single molecule fluorescent in situ hybridization (smFISH) approaches have been implemented to quantify mRNA decay rates in single, unperturbed cells...
2018: Methods in Molecular Biology
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