@article {Dobrzycki149591, author = {Tomasz Dobrzycki and Monika Krecsmarik and Florian Bonkhofer and Roger K. Patient and Rui Monteiro}, title = {An optimized pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridization}, elocation-id = {149591}, year = {2017}, doi = {10.1101/149591}, publisher = {Cold Spring Harbor Laboratory}, abstract = {Recent advances in genome engineering technologies 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 fish 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. Applying this approach to ISH, however, requires an efficient and robust 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 subsequent genotyping after ISH. In addition, we provide an image analysis protocol for quantifying gene expression in the trunks of ISH-probed embryos, easily adaptable to analyse 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 correlates well with 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.}, URL = {https://www.biorxiv.org/content/early/2017/06/13/149591}, eprint = {https://www.biorxiv.org/content/early/2017/06/13/149591.full.pdf}, journal = {bioRxiv} }