An optimised pipeline for parallel image-based quantification of gene expression and genotyping after <i>in situ</i> hybridisation.

ABSTRACT: 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 hybridisation (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

SUBMITTER: Dobrzycki T 

PROVIDER: S-EPMC5936060 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature