Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 155, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/59956
Keywords
Developmental Biology; Issue 155; genotyping; in situ hybridization; zebrafish; mutants; bias; image quantification
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Funding
- Wellcome Trust Chromosome and Developmental Biology PhD Scholarship [WT102345/Z/13/Z]
- British Heart Foundation (BHF IBSR Fellowship) [FS/13/50/30436]
- BHF Centre of Research Excellence, Oxford [RE/13/1/30181]
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In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.
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