PUBLICATION

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

Authors
Dobrzycki, T., Krecsmarik, M., Bonkhofer, F., Patient, R., Monteiro, R.
ID
ZDB-PUB-180315-2
Date
2018
Source
Biology Open   7(4): (Journal)
Registered Authors
Monteiro, Rui, Patient, Roger K.
Keywords
Bias, Genotyping, Image quantification, Mutant, Zebrafish
MeSH Terms
none
PubMed
29535102 Full text @ Biol. Open
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 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.
Genes / Markers
Marker Marker Type Name
dnmt3bb.1GENEDNA (cytosine-5-)-methyltransferase 3 beta, duplicate b.1
gata2aGENEGATA binding protein 2a
gpr65GENEG protein-coupled receptor 65
lmo4aGENELIM domain only 4a
runx1GENERUNX family transcription factor 1
tgfb3GENEtransforming growth factor, beta 3
tgfbr2bGENEtransforming growth factor beta receptor 2b
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Figures
Figure Gallery (5 images)
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Expression
Phenotype
Fish Conditions Stage Phenotype Figure
WT + MO2-gpr65standard conditionsPrim-5
lmo4auob100/uob100standard conditionsPrim-5
runx1hg1/hg1standard conditionsPrim-15
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Mutations / Transgenics
Allele Construct Type Affected Genomic Region
hg1
    		Point Mutation
    uob100
      		Small Deletion
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      Human Disease / Model
      No data available
      Sequence Targeting Reagents
      Target Reagent Reagent Type
      gpr65MO2-gpr65MRPHLNO
      lmo4aTALEN1-lmo4aTALEN
      1 - 2 of 2
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      Fish
      Fish
      lmo4auob100/+
      lmo4auob100/uob100
      runx1hg1/hg1
      runx1hg1/+
      WT
      WT + MO2-gpr65
      1 - 6 of 6
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      Antibodies
      Orthology
      Engineered Foreign Genes
      No data available
      Mapping
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      Citation
      Dobrzycki, T., Krecsmarik, M., Bonkhofer, F., Patient, R., Monteiro, R. (2018) An optimised pipeline for parallel image-based quantification of gene expression and genotyping after in situ hybridisation.. Biology Open. 7(4).