幽门螺杆菌基因组表达谱芯片的研制

Development of Helicobacter Pylori Genomic Expression Microarray

摘要:

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。

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.