The Research On Screening Differentially Expression Genes In Hirschsprung Disease By Using Microarray

Objective:To study the Differential expression of genes between Hirschsprung disease（HD）andnormal tissue by using microarray，to provide some guideline for the study of mechanismof HD development and establish the gene expression profiles of HD.Methods：In the period of January2011to January2012, we collected10specimens ofpathologically confirmed HD tissue and adjacent corresponding normal tissue fromdepartment of Surgery Guangdong Women and Children Hospital of Guangzhou MedicalCollege to extract total RNA.In this study，4colon tissues (4are HD tissues,4are theircorresponding normal tissues) were detected by the Agilent SurePrint G3Human GE8x60K Microarrays. Using SAM (significance analysis of microarray，SAM) softwareto analyse the signaling values of these genes and obtain Differential expression of genes.Scan for the fluorescent signals by using Agilent G3scanner and extract the date byFeature extraction software, then analyze differences in gene expression by importingdatas to Agilent genespring GX analytical software. we used Gene ontology (GO)system to classify the Differential expression of genes between HD and normal tissue andmake a bioinformatics analysis of them.Using RT-PCR to verify the Microarray test results.Results：According to signaling values≥800in the4specimens at least as the significant standard，we have selected a common expressed genes of12125. Using SAM software to analysethe signaling values of these genes and according to fold changes≥2.0times, or≤0.5times as the differentially significant standard,4pairs of specimens have a common Differential expression of genes of622, the expression of584（93.89%） of which weredown-regulated and the expression of38（6.11%） of which were up-regulated in the622genes. Cluster analysis of differentially expression genes showed that the HD tissues andthe normal tissues distinctly separate groups. Among the ten top ranked up-anddown-regulated genes (totally20), all of them were unknown differentially expressiongenes of HD. These genes were divided by molecular function(Molecular Function)according to GO (gene ontology) Category，the most of these genes are relevant tomuscle organ development,cell adhesion, cell-substrate junction assembly, calcium iontransport into cytosol,nervous system development,cytoskeleton organization, axonguidance, glial cell development et al.Test and verify6genes of all by RT-PCR, which expression direction consistent withthe results detected by Microarray, according with expected results.Conclusion:Using the microarray to screen the difference of gene expression profiles might be ofbenefit to rapid select the relative genes of the HD. There are obviously difference in thelevel of mRNA between HD tissue and normal tissue. The differentially expression genesin this study may refer to some important biological process in the development of HD.These genes may have some relationship with HD. We still need more study to verify thefunction of these genes in ontogenesis of HD. By this study, we can limit the related genesfor HD from mass of gene information. We can study the related function between thesegenes，find some candidate genes to study their change in gene or protein in thedevelopment of HD.