scholarly journals An optimized pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridization

2017 ◽  
Author(s):  
Tomasz Dobrzycki ◽  
Monika Krecsmarik ◽  
Florian Bonkhofer ◽  
Roger Patient ◽  
Rui Monteiro
Keyword(s):  
Gene Expression ◽  
Image Analysis ◽  
In Situ Hybridization ◽  
Quantitative Methods ◽  
Genome Engineering ◽  
Expression Patterns ◽  
Computer Based ◽  
Molecular Phenotypes ◽  
Mutant Lines

ABSTRACTAdvances in genome engineering have resulted in the generation of numerous zebrafish mutant lines. A commonly used method to assess gene expression in the mutants is in situ hybridization (ISH). Because the embryos can be distinguished by genotype after ISH, comparing gene expression between wild type and mutant siblings can be done blinded and in parallel. Such experimental design reduces the technical variation between samples and minimises the risk of bias. This approach, however, requires an efficient method of genomic DNA extraction from post-ISH fixed zebrafish samples to ascribe phenotype to genotype. Here we describe a method to obtain PCR-quality DNA from 95-100% of zebrafish embryos, suitable for genotyping after ISH. In addition, we provide an image analysis protocol for quantifying gene expression of ISH-probed embryos, adaptable for the analysis of different expression patterns. Finally, we show that intensity-based image analysis enables accurate representation of the variability of gene expression detected by ISH and that it can complement quantitative methods like qRT-PCR. By combining genotyping after ISH and computer-based image analysis, we have established a high-confidence, unbiased methodology to assign gene expression levels to specific genotypes, and applied it to the analysis of molecular phenotypes of newly generated lmo4a mutants.SUMMARY STATEMENTOur optimized protocol to genotype zebrafish mutant embryos after in situ hybridization and digitally quantify the in situ signal will help to standardize existing experimental designs and methods of analysis.

scholarly journals RNA In Situ Hybridization for Detecting Gene Expression Patterns in the Abdomens and Wings of Drosophila Species

2021 ◽  
Vol 4 (1) ◽  
pp. 20
Author(s):  
Mujeeb Shittu ◽  
Tessa Steenwinkel ◽  
William Dion ◽  
Nathan Ostlund ◽  
Komal Raja ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Drosophila Species ◽  
Gene Expression Patterns ◽  
Probe Design ◽  
Tissue Preparation ◽  
Design And Synthesis ◽  
Rna In Situ Hybridization

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

scholarly journals Spatially multiplexed RNA in situ hybridization to reveal tumor heterogeneity

2019 ◽  
Vol 48 (3) ◽  
pp. e17-e17 ◽  
Author(s):  
Lena Voith von Voithenberg ◽  
Anna Fomitcheva Khartchenko ◽  
Deborah Huber ◽  
Peter Schraml ◽  
Govind V Kaigala
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Spatial Heterogeneity ◽  
Tumor Heterogeneity ◽  
Expression Patterns ◽  
Spatial Multiplexing ◽  
Local Analysis ◽  
Tissue Sections ◽  
Rna In Situ Hybridization

Abstract Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.

scholarly journals How to distinguish between different cell lineages sharing common markers using combinations of double in-situ-hybridization and immunostaining in avian embryos: CXCR4-positive mesodermal and neural crest-derived cells

2020 ◽  
Vol 155 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Imadeldin Yahya ◽  
Marion Böing ◽  
Beate Brand-Saberi ◽  
Gabriela Morosan-Puopolo
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Embryonic Development ◽  
Neural Crest ◽  
Expression Patterns ◽  
Simultaneous Detection ◽  
Neural Crest Cells ◽  
Cell Lineages ◽  
Wide Range

AbstractCell migration plays a crucial role in early embryonic development. The chemokine receptor CXCR4 has been reported to guide migration of neural crest cells (NCCs) to form the dorsal root ganglia (DRG) and sympathetic ganglia (SG). CXCR4 also plays an important part during the formation of limb and cloacal muscles. NCCs migration and muscle formation during embryonic development are usually considered separately, although both cell lineages migrate in close neighbourhood and have markers in common. In this study, we present a new method for the simultaneous detection of CXCR4, mesodermal markers and NCCs markers during chicken embryo developmental stages HH18–HH25 by combining double whole-mount in situ hybridization (ISH) and immunostaining on floating vibratome sections. The simultaneous detection of CXCR4 and markers for the mesodermal and neural crest cells in multiple labelling allowed us to compare complex gene expression patterns and it could be easily used for a wide range of gene expression pattern analyses of other chicken embryonic tissues. All steps of the procedure, including the preparation of probes and embryos, prehybridization, hybridization, visualization of the double labelled transcripts and immunostaining, are described in detail.

scholarly journals A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

2017 ◽  
Author(s):  
Chiara Sinigaglia ◽  
Daniel Thiel ◽  
Andreas Hejnol ◽  
Evelyn Houliston ◽  
Lucas Leclère
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Developmental Regulation ◽  
Expression Patterns ◽  
Urea Solution ◽  
Similar Reduction ◽  
In Situ Detection ◽  
Precise Expression ◽  
Significant Health

AbstractIn situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has proven to be essential to our understanding of biological processes, including developmental regulation. In situ protocols are today routine in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acids strand. This step, in general, is carried out at high temperatures and in a denaturing solution, the hybridization buffer, commonly containing 50% (v/v) formamide. An important drawback is that hot formamide poses a significant health risk and so must be handled with great care.We were prompted to test alternative hybridization solutions for in situ detection of gene expression in the medusa of the hydrozoan Clytia hemisphaerica, where traditional protocols caused extensive deterioration of the morphology and texture during hybridization, hindering observation and interpretation of results. Inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8 M urea solution in the hybridization buffer. The new protocol yielded better morphologies and consistency of tissues, and also notably improved the resolution of the signal, allowing more precise localization of gene expression, as well as reduced staining at non-specific sites. Given the improved results using a less toxic hybridization solution, we tested the urea protocol on a number of other metazoans: two brachiopod species (Novocrania anomala and Terebratalia transversa) and the worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea in in situ hybridization offers safer alternative protocols, potentially useful in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

ZebraFISH: Fluorescent In Situ Hybridization Protocol and Three-Dimensional Imaging of Gene Expression Patterns

Zebrafish ◽  
2006 ◽  
Vol 3 (4) ◽  
pp. 465-476 ◽  
Author(s):  
Monique C.M. Welten ◽  
Simon B. de Haan ◽  
Niels van den Boogert ◽  
Jasprien N. Noordermeer ◽  
Gerda E.M. Lamers ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Fluorescent In Situ Hybridization ◽  
Expression Patterns ◽  
Three Dimensional ◽  
Gene Expression Patterns ◽  
Three Dimensional Imaging ◽  
Hybridization Protocol

Determination of gene expression patterns using in situ hybridization to Drosophila testes

2009 ◽  
Vol 4 (12) ◽  
pp. 1807-1819 ◽  
Author(s):  
Ceri A Morris ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Expression Patterns ◽  
Gene Expression Patterns
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