Highly Multiplexed Subcellular RNA Sequencing in Situ

Science (Impact Factor: 33.61). 02/2014; 343(6177). DOI: 10.1126/science.1250212
Source: PubMed


Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8742 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

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    • "This method uses a RNA capture procedure that is both noninvasive and spatially precise with potential applications in embryo, neuron and cancer cell studies where the understanding of the very specific spatiotemporal gene expression pattern is important [3]. Another method, Fluorescent In Situ RNA Sequencing (FISSEQ), allows highly multiplexed subcellular RNA sequencing in situ by stably cross-linking cDNA amplicons, which are sequenced within a biological sample [44]. These technologies are extremely exciting allowing us to study individual cells for their transcriptomics in their natural microenvironment. "
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    ABSTRACT: Single-cell analysis heralds a new era that allows "omics" analysis, notably genomics, transcriptomics, epigenomics and proteomics at the single-cell level. It enables the identification of the minor subpopulations that may play a critical role in a biological process of a population of cells, which conventionally are regarded as homogeneous. It provides an ultra-sensitive tool to clarify specific molecular mechanisms and pathways and reveal the nature of cell heterogeneity. It also facilitates the clinical investigation of patients when a very low quantity or a single cell is available for analysis, such as noninvasive prenatal diagnosis and cancer screening, and genetic evaluation for in vitro fertilization. Within a few short years, single-cell analysis, especially whole genomic sequencing and transcriptomic sequencing, is becoming robust and broadly accessible, although not yet a routine practice. Here, with single cell RNA-seq emphasized, an overview of the discipline, progresses, and prospects of single-cell analysis and its applications in biology and medicine are given with a series of logic and theoretical considerations.
    04/2014; 3(1). DOI:10.4172/2168-9431.1000106
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    • "New insights into the molecular basis of such diversity and the mechanism of mRNP-specific assembly/disassembly at the single-molecule level awaits the invention of new technologies that will allow us to investigate protein composition of single mRNP granules. Combining such techniques with fluorescent in situ RNA sequencing (FISSEQ) (Lee et al., 2014), which visualizes thousands of different RNA molecules within a cell in situ, is an exciting avenue that will lead to the molecular dissection of individual mRNP granules. These results provide a tantalizing intersection of two major themes from the issue as insight into why Location Matters may ultimately emerge from studies harnessing The Power of One—analysis of single mRNAs and mRNPs. "
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    ABSTRACT: The subcellular position of a protein is a key determinant of its function. Mounting evidence indicates that RNA localization, where specific mRNAs are transported subcellularly and subsequently translated in response to localized signals, is an evolutionarily conserved mechanism to control protein localization. On-site synthesis confers novel signaling properties to a protein and helps to maintain local proteome homeostasis. Local translation plays particularly important roles in distal neuronal compartments, and dysregulated RNA localization and translation cause defects in neuronal wiring and survival. Here, we discuss key findings in this area and possible implications of this adaptable and swift mechanism for spatial control of gene function.
    Cell 03/2014; 157(1):26-40. DOI:10.1016/j.cell.2014.03.005 · 32.24 Impact Factor
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    ABSTRACT: New methods employ RNA-seq to study single cells within complex tissues by in situ sequencing or mRNA capture from single photoactivated cells.
    Genome Biology 03/2014; 15(3):110. DOI:10.1186/gb4169 · 10.81 Impact Factor
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