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Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos.

Development (Cambridge, England) (2015-12-25)
L Carine Stapel, Benoit Lombardot, Coleman Broaddus, Dagmar Kainmueller, Florian Jug, Eugene W Myers, Nadine L Vastenhouw
ABSTRACT

Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines have hampered quantification at the (sub)cellular level in complex samples such as tissues and embryos. Here, we present a protocol for smFISH on zebrafish embryo sections in combination with an image analysis pipeline for automated transcript detection and cell segmentation. We use this strategy to quantify gene expression differences between different cell types and identify differences in subcellular transcript localization between genes. The combination of our smFISH protocol and custom-made, freely available, analysis pipeline will enable researchers to fully exploit the benefits of quantitative transcript analysis at cellular and subcellular resolution in tissues and embryos.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Poly-L-lysine solution, 0.1 % (w/v) in H2O
Sigma-Aldrich
Glucose Oxidase from Aspergillus niger, Type VII, lyophilized powder, ≥100,000 units/g solid (without added oxygen)
Sigma-Aldrich
Catalase from Aspergillus niger, ammonium sulfate suspension, ≥4,000 units/mg protein
Sigma-Aldrich
Dextran sulfate sodium salt from Leuconostoc spp., for molecular biology, average Mw >500,000 (dextran starting material), contains 0.5-2% phosphate buffer