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

Research output: Contribution to journalArticlepeer-review


  • Tomasz Dobrzycki
  • Monika Krecsmarik
  • Florian Bonkhofer
  • Roger Patient
  • Rui Monteiro

Colleges, School and Institutes

External organisations

  • University of Oxford


Advances 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.


Original languageEnglish
Pages (from-to)bio.031096
JournalBiology Open
Publication statusPublished - 13 Mar 2018


  • zebrafish , mutant , bias , genotyping , image qualification