两种标记方法对60mer寡核苷酸芯片背景信号影响的初步分析  

Evaluation of Two Different Sample Labeling Methods on Background Signal Intensities for 60 mer Oligonucleotide Microarrays

作　　者：张亚莉[1] 马文丽[1] 莫小阳[1] 石嵘[1] 李凌[1] 徐秋林[1] 张海燕[1] 郑文岭[1] 

机构地区：[1]南方医科大学基因工程研究所,广州510515

出　　处：《中国生物工程杂志》2006年第1期42-45,共4页China Biotechnology

摘　　要：研究两种不同的样本标记方法对人全基因组高密度60mer寡核苷酸芯片背景信号的影响。收集5对患病与健康人外周血单个核细胞,分别提取总RNA后,采用限制性显示技术(restrictiondisplay,RD)进行样本双色(Cy3/Cy5)荧光标记,与5张Agilent60mer高密度(22K)Human1B寡核苷酸芯片进行杂交。芯片全部杂交点分3组基因探针组、阳性对照组和阴性对照组。阳性对照采用荧光标记寡核苷酸直接掺入法进行标记。对全部杂交信号点的Cy3和Cy5背景信号值,用SPSS软件进行数据转换、正态性检验、方差齐性检验、变异系数分析和重复数据的方差分析。数据分析结果显示,Cy3标记的背景信号值均高于Cy5标记的背景信号值。重复测量数据的方差分析表明,在Cy3和Cy5标记中,两种不同标记方法间的背景信号值的差异极显著(PCy3<0.01,PCy5<0.01),且RD标记点的背景信号平均值低于荧光标记寡核苷酸直接掺入标记法标记的阳性对照点。RD标记方法是一种有用的低背景信号的高密度长链寡核苷酸芯片样本标记方法。The effects of two different sample labeling methods on background signal intensities for highdensity 60mer oligonucleotide microarray were investigated. Peripheral blood samples from five disease and five control subjects were collected. Total RNA targets from peripheral blood mononuclear ceils were extracted and labeled with RD-PCR protocol, which were hybridized to Agilent Human 1B oligonucleotide microarrays in a twocolor comparative format. The positive control targets were labeled with the directly incorporated fluorescentlylabeled dNTP labeling. The SPSS program was performed to test normality of the dataset, variance homogeneity between the groups, coefficients of variation （CV） and analysis of variance. The results showed that the background signal intensities of Cy3 channel were higher than those of Cy5 channel. The differences of dNTP labeling were extremely significant （Pcy3 〈 0.01, Pcy5 〈 0.01 ）. It is concluded that the RD-PCR is a potential and useful sample labeling method with lower background signal intensities for studying high-density long oligonucleotide microarray.

关 键 词：限制性显示技术 寡核苷酸芯片 背景信号强度 

分 类 号：Q819[生物学—生物工程]