一种用于基因表达谱分析的基因芯片方法

A Microarray Method for Analyzing Gene Expression Profiles

目的通过研究SLE患者外周血基因表达谱的改变 ,建立一种新型的基因芯片技术用于分子发病机理的研究。 方法提取总RNA ,逆转录合成单链cDNA、双链cDNA ,体外转录合成生物素标记的cRNA与基因芯片进行杂交 ,通过抗体的检测标记荧光染料Cy3,基因芯片扫描仪进行图像扫描。 结果该方法有较高的重复性和稳定性 ,SLE患者与正常对照相比较 ,鉴定出 94个基因存在表达差异。 结论该方法能在较少起始标本量的情况下 ,有效的进行SLE致病基因的筛选 ,更好的理解SLE发生的分子机理 。

Objective To establish a microarray method for investigating molecular pathogenesis of rheumatoid diseases through studying the difference in gene expression of peripheral white blood cells between SLE patients and healthy controls. Methods Total RNA was extracted from the peripheral white blood cells of normal human and systemic lupus erythematosus double strand cDNA template was synthesed from total RNA and cRNA probe was transcribed with biotin labeling. After hybridization of probe with microarray, binding of streptavidin to biotin was performed to be amplificated with first antibody, and further amplificated with Cy3-Conjugated second antibody. Then detection of Cy3 dye was carried with ScanArray 5000. Results Date were analyzed to select over- or under-expressed genes in SLE patients versus controls. Among over 3000 target genes, 94 genes were identified in SLE patients that were either over- or under-expressed compared to controls, 33 genes were up-regulated and 61 genes down-regulated. Conclusion These results successfully demonstrate the use of the new microarray technique is effective in screening the differentially expressed genes of SLE and better understanding the pathogenesis of SLE. This microarray techique may apply to other disease as well.