幽门螺杆菌基因组表达谱芯片的研制

Development of Helicobacter Pylori Genomic Expression Microarray

以H．pylori 26695和J99作为模板,采用多聚酶链反应(PCR)扩增得到所需要的开放阅读框(open read- ing frame,ORF)片段。使用Genemachine点样仪进行点样,采用随机九聚体、Cy3-dCTP或Cy5-dCTP和Super- scriptⅡ标记H．Pylori RNA标本,完成芯片杂交过程(?)数据判断标准是标化后Cy3/Cy5比值(ratio)≤0．5为基因表达上调,若ratio值≥2．0则为表达下调。采用芯片重复性、信噪比评估芯片质量,应用RT-PCR反应验证芯片结果。研制的H．pylori基因组DNA芯片共包括1636个ORF,其中H．pylori-26695为1549个,J99为87个。表达谱数据分析显示大多数阵列具有显著高于背景的杂交信号,信噪比(S/N)≥2的基因点数约占总基因数的87．76%。芯片内点间重复率约为94．05%。通过RT-PCR反应显示,在所选择的20个基因中,大部分基因的RT-PCR结果与芯片检测结果一致。

The open reading frame （ORF） fragments on our microarray were generated by polymerase chain reaction （PCR） using genespecific primers. Genomic DNA of H. pylori 26695 and J99 ware used as templates. DNA fragments on the array were printed by Genemachine printor. Using random nanomer, Cy3-dCTP/CyS-dCTP and Superscript Ⅱ to 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-regulated, 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 botwecn 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 RT-PCR.