Blog
About

  • Record: found
  • Abstract: found
  • Article: not found

Programmable in situ amplification for multiplexed imaging of mRNA expression

Read this article at

ScienceOpenPublisherPMC
Bookmark
      There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

      Abstract

      In situ hybridization methods enable the mapping of mRNA expression within intact biological samples 1,2. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos 36 – a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR) 7. Using this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit excellent sample penetration, high signal-to-background, and sharp signal localization.

      Related collections

      Most cited references 40

      • Record: found
      • Abstract: found
      • Article: not found

      Imaging individual mRNA molecules using multiple singly labeled probes

      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 via fluorescence microscopy. We demonstrate simultaneous detection of three mRNA species in single cells and mRNA detection in yeast, nematodes, fruit fly wing discs, mammalian cell lines and neurons.
        Bookmark
        • Record: found
        • Abstract: found
        • Article: not found

        A DNA-fuelled molecular machine made of DNA.

        Molecular recognition between complementary strands of DNA allows construction on a nanometre length scale. For example, DNA tags may be used to organize the assembly of colloidal particles, and DNA templates can direct the growth of semiconductor nanocrystals and metal wires. As a structural material in its own right, DNA can be used to make ordered static arrays of tiles, linked rings and polyhedra. The construction of active devices is also possible--for example, a nanomechanical switch, whose conformation is changed by inducing a transition in the chirality of the DNA double helix. Melting of chemically modified DNA has been induced by optical absorption, and conformational changes caused by the binding of oligonucleotides or other small groups have been shown to change the enzymatic activity of ribozymes. Here we report the construction of a DNA machine in which the DNA is used not only as a structural material, but also as 'fuel'. The machine, made from three strands of DNA, has the form of a pair of tweezers. It may be closed and opened by addition of auxiliary strands of 'fuel' DNA; each cycle produces a duplex DNA waste product.
          Bookmark
          • Record: found
          • Abstract: found
          • Article: not found

          NUPACK: Analysis and design of nucleic acid systems.

          The Nucleic Acid Package (NUPACK) is a growing software suite for the analysis and design of nucleic acid systems. The NUPACK web server (http://www.nupack.org) currently enables: thermodynamic analysis of dilute solutions of interacting nucleic acid strands. sequence design for complexes of nucleic acid strands intended to adopt a target secondary structure at equilibrium.Utilities: evaluation, display, and annotation of equilibrium properties of a complex of nucleic acid strands. NUPACK algorithms are formulated in terms of nucleic acid secondary structure. In most cases, pseudoknots are excluded from the structural ensemble.
            Bookmark

            Author and article information

            Affiliations
            [1] Department of Bioengineering, California Institute of Technology, Pasadena, CA 91125, USA
            [2] Department of Biology, California Institute of Technology, Pasadena, CA 91125, USA
            [3] Department of Applied & Computational Mathematics, California Institute of Technology, Pasadena, CA 91125, USA
            Author notes
            Journal
            9604648
            20305
            Nat Biotechnol
            Nature biotechnology
            1087-0156
            1546-1696
            12 October 2010
            31 October 2010
            November 2010
            1 May 2011
            : 28
            : 11
            : 1208-1212
            3058322
            21037591
            10.1038/nbt.1692
            nihpa240064

            Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

            Funding
            Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: R01 EB006192-04 ||EB
            Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: R01 EB006192-03 ||EB
            Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: R01 EB006192-02 ||EB
            Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: R01 EB006192-01 ||EB
            Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: P50 HG004071-04 ||HG
            Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: P50 HG004071-03 ||HG
            Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: P50 HG004071-02 ||HG
            Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
            Funded by: National Human Genome Research Institute : NHGRI
            Award ID: P50 HG004071-01 ||HG
            Categories
            Article

            Biotechnology

            Comments

            Comment on this article