Programmable in situ amplification for multiplexed imaging of mRNA expression

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing 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). With 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 deep sample penetration, high signal-to-background ratios and sharp signal localization

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Multiplexed Quantitative In Situ Hybridization for Mammalian Cells on a Slide: qHCR and dHCR Imaging (v3.0)

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables multiplexed quantitative mRNA imaging in diverse sample types. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports two quantitative imaging modes: (1) qHCR imaging for analog mRNA relative quantitation with subcellular resolution and (2) dHCR imaging for digital mRNA absolute quantitation with single-molecule resolution. Here, we provide protocols for qHCR and dHCR imaging in mammalian cells on a slide

DNA Nanotechnology

Cite this entry as: Yaradoddi J.S. et al. (2019) DNA Nanotechnology. In: Martínez L., Kharissova O., Kharisov B. (eds) Handbook of Ecomaterials. Springer, Cham DOI: https://doi.org/10.1007/978-3-319-68255-6_191 First Online: 14 February 2019 Online ISBN: 978-3-319-68255-6 Print ISBN: 978-3-319-68254-9Since from the past few decades DNA appeared as an excellent molecular building block for the synthesis of nanostructures because of its probable encoded and confirmation intra- and intermolecular base pairing, various case strategies and consistent assembly techniques have been established to manipulate DNA nanostructures to at higher complexity. The capability to develop DNA construction with precise special control has permitted scientists to discover novel applications in many ways, such as scaffold development, sensing applications, nanodevices, computational applications, nanorobotics, nanoelectronics, biomolecular catalysis, disease diagnosis, and drug delivery. The present chapter emphasizes to brief the opportunities, challenges, and future prospective on DNA nanotechnology and its advancements.Peer reviewe

Highly specific multiplexed RNA imaging in tissues with split-FISH

10.1038/s41592-020-0858-0NATURE METHODS17