Development of Helicobacter Pylori Genomic Expression Microarray

Abstract:

The open reading frame(ORF)fragments on our microarray were generated by polymerase chain reaction(PCR)using gene- specific primers.Genomic DNA of H.pylori 26695 and J99 ware used as templates.DNA fragments on the array were printed by Genema- chine printor.Using random nanomer,Cy3-dCTP/Cy5-dCTP and SuperscriptⅡto label H.pylori RNA and complete hybridization.Results were judged on the basis of normalized Cy3/Cy5 ratio value,that is,genes with ratio less than or equal to 0.5 were considered down-regula- ted,those with ratio greater than or equal to 2.0 were up-regulated The quality of microarray was evaluated by means of reproducibility and signal/noise ratio. Microarray results were tested by RT-PCR. The final microarray included 1636 ORFs of both strains. Repetitive rate be-tween different dots within the same microarray was 94.05%.Most array had significantly higher signal than background,with 87.76% spots had signal/noise greater than or equal to 2. Most genes from 20 genes selected for testing microarray results were verified by TR-PCR.