Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Summary

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Abstract

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.

Protocol

Part 1: Fluorescent Labeling of cDNA The Cy3 dye used in the microarray experiment is sensitive to photo-degradation. The procedure should take place in a minimal amount of light. The labeling protocol was adapted from the BioPrime Array CGH Genomic Labeling System Manual1. The reagents for the labeling reaction are stored at -20°C. From the BioPrime Array CGH Genomic Labeling System thaw 2.5X Random Primer Buffer, 10X dCTP mix, and nuclease-free water. Also thaw the C…

Discussion

Many microarray protocols, including the one detailed here, use fluorescent dyes as a means of measuring and quantifying hybridization. The Cy3 fluorescent dye is sensitive to photo and ozone degradation. It is recommended that the procedure be conducted in minimal lighting. In addition, small dust particles can interfere with hybridization and scanning. Special attention should be given to make sure the work area is clean and free from dust.

There are many alternatives to the labeling protoc…

Materials

Material Name	Type	Company	Catalogue Number	Comment
Array Microcentrifuge Dryer		ISC Bioexpress	C-1303-T
BioPrime Array CGH Genomic Labeling System		Invitrogen	18095-011
Cyanine 3-dCTP		PerkinElmer	NEL576
GenePix 4000B		Molecular Devices	GENEPIX 4000B
Microcon YM-30		Millipore	42411
NimbleGen Array Processing Accessories		Roche NimbleGen	5223539001
NimbleGen Hybridization Kit		Roche NimbleGen	5223474001
NimbleGen Hybridization System 4		Roche NimbleGen	5223652001
NimbleGen Wash Buffer Kit		Roche NimbleGen	5223504001
X1 Mixers		Roche NimbleGen	5223725001