基于荧光微球液相基因表达阵列的建立  

Development of a Bead-based Liquid Array for Analysis of Gene Expression Profiling

作　　者：邵雪君[1,2] 陈子兴[1] 缪美华[2] 岑建农[1] 沈宏杰[1] 

机构地区：[1]苏州大学附属第一人民医院江苏省血液研究所,苏州215006 [2]苏州大学附属儿童医院检验科,苏州215003

出　　处：《生物化学与生物物理进展》2011年第7期661-669,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(30470733)~~

摘　　要：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.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 establish. Multiplex ligation-dependent probe amplification （MLPA） was employed to amplify several targeted cDNAs using only one pair identical primers, each MLPA probe consisted of two short synthetic oligonucleotides, and the tag which was coupled chemically to the fluorescent beads was complementary to one probe. Five beads with different fluorescence intensity coupled to RAC2, RhoBTB3, SPA-1, Rap 1GAP and GAPDH were established. Biotinylated PCR amplicons were then hybridized to the compIementary tag on each bead set. Bound amplicons were detected by flow cytometry using a streptavidin-linked reporter dye, PE. 111 BM specimens were analyzed in total, include RA（22）, RAEB（22）, RAEBt（9）, AML（33）, and control group （22, including hyperplastic anemia, iron deficiency anemia, aplastic anemia et al）, and the difference of the transcriptional level of RAC2, RhoBTB3, SPA-1 and Rap I GAP relative to GAPDH were analyzed using wilcoxon non-parametric test and SNK method among different groups. The results were confirmed by RQ-PCR. The bead-based fiow-cytometric array had an excellent sensitivity and a wide linear range, could get a positive signal for PCR product from 0.002 5 to 0.1 μmol, the fine specificity was proved by no cross-hybridization signals presented among different bead set, and the reproducibility were also good enough（P 〈 0.001）. The expression profiling of RAC2, RhoBTB3, SPA-1, RaplGAP and GAPDH detected by this liquid bead-based flow-cytometric array were obtained,

关 键 词：基因表达谱 微球 连接 流式细胞 杂交 

分 类 号：Q78[生物学—分子生物学]