基于荧光微球液相基因表达阵列的建立

建立基于荧光微球的液相基因表达阵列,用于特定基因组合的表达谱分析.采用带有不同强度荧光鉴别信号的羧基化微球,与氨基修饰的不同标签寡核苷酸序列化学偶联,制成微球阵列.多重连接依赖的探针扩增技术(MLPA)用于扩增靶基因核苷酸序列,即通过RNA标本六随机引物逆转成cDNA,与不同基因特异性的一对探针杂交,耐高温的连接酶联接,最后采用生物素标记的同一对引物扩增.PCR产物与微球阵列液相杂交,加入链亲和素标记的PE染料,上流式细胞仪检测.应用这一系统检测骨髓增生异常综合症中难治性贫血(RA)、难治性贫血伴原始细胞增多(RAEB)、难治性贫血伴转化中原始细胞增多(RAEBt)、急性髓细胞性白血病(AML)和其他组(包括再生障碍性贫血、血小板减少、巨幼贫、溶贫等)差异表达谱,差异表达结果用实时荧光定量PCR验证.共建立了5个基因的微球阵列,分别为Rap1GAP、RAC2、SPA1、RhoBTB3和内参GAPDH,每个基因检测的线性范围为0.002 5～0.1μmol,液相表达阵列具有良好的特异性和重复性(P<0.001).检测RA、RAEB、RAEBt、AML和其他组差异表达发现,RAC2、RhoBTB3、SPA-1和Rap1GAP各组间有显著性差异性存在(分别为P<0.000 1,P=0.049 1,P=0.020 6和P=0.004 6),其差异显著性与实时荧光定量PCR一致,泊松相关系数分别为0.930,0.946,0.945和0.921,具显著性(P<0.001).结果表明,成功建立了基于荧光微球的液相基因表达阵列,其敏感性高、特异性强、重复性好.

Development of a bead-based liquid array for analysis of gene expression profiling

It is well known that the gene expression profiling can be detected by RT-PCR singly,or which can be detected by cDNA array in large numbers,however,to evaluate the expression of several targeted genes in a special regulation pathway,or some interested genes in a certain disease simultaneously,the methods were limited.So,development of a simple,robust,sensitive,specific and economic assay satisfied the need mentioned above was very useful,and a bead-based flow-cytometric multiplex assay was aimed to establi...