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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.
A liquid-phase gene expression array based on fluorescent microspheres was established for expression profiling of specific gene combinations.Carboxylated microspheres with different intensity fluorescence discrimination signals were used to chemically couple with amino-tagged different tagged oligonucleotide sequences (MLPA) is used to amplify the nucleotide sequence of the target gene, ie, by reverse transcription of the RNA sample by six random primers into cDNA, and with a pair of probes of different gene specificities Needle hybridization and high temperature resistant ligase, and finally amplified with the same pair of primers labeled with biotin.The PCR products were hybridized with the microsphere array liquid phase, added with streptavidin-labeled PE dye and detected by flow cytometry A systematic examination of refractory anemia (RA), refractory anemia with blasts blasts (RAEB), refractory anemia with transformed blasts of blasts (RAEBt), acute myeloid leukemia (AML) ) And other groups (including aplastic anemia, thrombocytopenia, megaloblastic and Poverty-poor, etc.), and the differential expression results were verified by real-time fluorescence quantitative PCR.A total of 5 microspheres were constructed, which were Rap1GAP RAC2, SPA1, RhoBTB3 and internal reference GAPDH, the linear range of each gene was 0.002 5 ~ 0.1μmol, and the array of liquid phase showed good specificity and repeatability (P <0.001) In the other groups, there were significant differences among RAC2, RhoBTB3, SPA-1 and Rap1GAP groups (P <0.0001, P = 0.0491, P = 0.0206 and P = 0.004 6, respectively) Significantly, the correlation coefficient of Poisson was 0.930, 0.946, 0.945 and 0.921, respectively (P <0.001), which was consistent with that of real-time fluorescence quantitative PCR.The results showed that the liquid phase gene expression array based on fluorescent microspheres Sensitive, specific, reproducible.