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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Available from: Je Hyuk Lee, Oct 25, 2014
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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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    • "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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