scholarly journals Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos

2017 ◽  
Author(s):  
Vikas Trivedi ◽  
Harry M.T. Choi ◽  
Scott E. Fraser ◽  
Niles A. Pierce
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Mrna Expression ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Developmental Gene Expression ◽  
Vertebrate Embryos ◽  
Anatomical Space ◽  
Subcellular Resolution

ABSTRACTFor decades, in situ hybridization methods have been essential tools for studies of vertebrate development and disease, as they enable qualitative analyses of mRNA expression in an anatomical context. Quantitative mRNA analyses typically sacrifice the anatomy, relying on embryo microdissection, dissociation, cell sorting, and/or homogenization. Here, we eliminate the tradeoff between quantitation and anatomical context, using multiplexed in situ hybridization chain reaction (HCR) to perform accurate and precise relative quantitation of mRNA expression with subcellular resolution within whole-mount vertebrate embryos. Gene expression can be queried in two directions: read-out from anatomical space to expression space reveals co-expression relationships in selected regions of the specimen; conversely, read-in from multidimensional expression space to anatomical space reveals those anatomical locations in which selected gene co-expression relationships occur. As we demonstrate by examining gene circuits underlying somitogenesis, quantitative read-out and read-in analyses provide the strengths of flow cytometry expression analyses, but by preserving subcellular anatomical context, they enable iterative bi-directional queries that open a new era for in situ hybridization.SUMMARYMultiplexed in situ hybridization chain reaction (HCR) enables quantitative multidimensional analyses of developmental gene expression with subcellular resolution in an anatomical context.

scholarly journals Combined in situ hybridization chain reaction and immunostaining to visualize gene expression in whole-mount Drosophila central nervous systems

2021 ◽  
Author(s):  
Julia C Duckhorn ◽  
Ian P Junker ◽  
Yun Ding ◽  
Troy R Shirangi
Keyword(s):  
Gene Expression ◽  
Nervous System ◽  
In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Nervous Systems ◽  
Whole Mount ◽  
Central Nervous Systems ◽  
Mrna And Protein Expression

Methods to visualize gene expression in the Drosophila central nervous system are important in fly neurogenetic studies. In this chapter, we describe a detailed protocol that sequentially combines in situ hybridization chain reaction (HCR) and immunostaining to detect mRNA and protein expression in whole-mount Drosophila larval and adult central nervous systems. We demonstrate the application of in situ HCR in comparisons of nervous system gene expression between Drosophila species, and in the validation of single-cell RNA-Seq results in the fly nervous system. Our protocol provides a simple, robust, multiplexable, and relatively affordable means to quantitatively visualize gene expression in the nervous system of flies, facilitating its general use in fly neurogenetic studies.

scholarly journals Combined microRNA and mRNA detection in mammalian retinas by in situ hybridization chain reaction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Pei Zhuang ◽  
Huanqing Zhang ◽  
Ryan M. Welchko ◽  
Robert C. Thompson ◽  
Shunbin Xu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Mrna Detection

HCR RNA-FISH protocol for the whole-mount brains of Drosophila and other insects v1

2021 ◽  
Author(s):  
Amanda A. G. Ferreira ◽  
Bogdan Sieriebriennikov ◽  
Hunter Whitbeck
Keyword(s):  
Drosophila Melanogaster ◽  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Alexa Fluor ◽  
Rna Fish ◽  
Whole Mount

This is a protocol to perform RNA fluorescent in situ hybridization (RNA-FISH) using hybridization chain reaction (HCR) on whole-mount samples of the brains of the fly Drosophila melanogaster and other insects, e.g. the jumping ant Harpegnathos saltator. Probes and HCR reagents are purchased from Molecular Instruments. This protocol is loosely based on the "generic sample in solution" protocol published by Molecular Instruments. Our modifications include the description of fixation conditions, counterstaining by Hoechst, and altered washes. Additionally, we use larger concentrations of probes and hairpins following the protocol described by Younger, Herre et al. 2020. We have successfully employed this protocol to stain insect brains with up to 4 different probe sets simultaneously (hairpins conjugated with Alexa Fluor 488, 546, 496, and 647).

scholarly journals Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for detection of microbes in sediments

2021 ◽  
Author(s):  
Zeyu Jia ◽  
Yijing Dong ◽  
Heng Xu ◽  
Fengping Wang
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
False Positive ◽  
Signal Intensity ◽  
Sample Pretreatment ◽  
Extraction Methods ◽  
Hybridization Chain Reaction ◽  
Chain Reaction ◽  
Widespread Application

AbstractFluorescence in situ hybridization (FISH) is a canonical tool commonly used in environmental microbiology research to visualize targeted cells. However, the problems of low signal intensity and false-positive signals impede its widespread application. Alternatively, the signal intensity can be amplified by incorporating Hybridization Chain Reaction (HCR) with FISH, while the specificity can be improved through protocol modification and proper counterstaining. Here we optimized the HCR-FISH protocol for studying microbes in environmental samples, particularly marine sediments. Firstly, five sets of HCR initiator/amplifier pairs were tested on the laboratory-cultured bacterium Escherichia coli and the archaeon Methanococcoides methylutens, and two sets displayed high hybridization efficiency and specificity. Secondly, we tried to find the best combination of sample pretreatment methods and HCR-FISH protocol for environmental sample analysis with the aim of producing less false positive signals. Various detachment methods, extraction methods and formulas of hybridization buffer were tested using sediment samples. Thirdly, an image processing method was developed to enhance the DAPI signal of microbial cells against that of abiotic particles, providing a reliable reference for FISH imaging. In summary, our optimized HCR-FISH protocol showed promise to serve as an addendum to traditional FISH for research on environmental microbes.

scholarly journals Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid Pseudomonas aeruginosa Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Peter Jorth ◽  
Melanie A. Spero ◽  
J. Livingston ◽  
Dianne K. Newman
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Single Cell ◽  
Spatial Scales ◽  
Hybridization Chain Reaction ◽  
Microbial Activities ◽  
Chain Reaction ◽  
Content Type ◽  
Hypoxic Zone

ABSTRACT It is well appreciated that oxygen- and other nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scales in situ. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to provide analog mRNA relative quantitation of Pseudomonas aeruginosa single cells as a step toward this end. To assess the potential for this method to be applied to bacterial populations, we visualized the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG) at single-cell resolution within laboratory-grown aggregates. After validating new HCR probes, we quantified algD and narG expression across microenvironmental gradients within both single aggregates and aggregate populations using the agar block biofilm assay (ABBA). For mucoid and nonmucoid ABBA populations, narG was expressed in hypoxic and anoxic regions, while alginate expression was restricted to the hypoxic zone (∼40 to 200 μM O2). Within individual aggregates, surface-adjacent cells expressed alginate genes at higher levels than interior cells, revealing that alginate expression is not constitutive in mucoid P. aeruginosa but instead varies with oxygen availability. These results establish HCR v3.0 as a versatile and robust tool to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. This advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen activities during infections. IMPORTANCE A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression in situ at single-cell resolution in bacterial aggregates. Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitro P. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.

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