Mo Xiao-yang, Ma Wen-li, Li Ling, Shi Rong, Zhang Bao, Xu Qiu-lin, Zhang Hai-yan, Zheng Wen-ling
Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China.
Di Yi Jun Yi Da Xue Xue Bao. 2005 Sep;25(9):1081-5, 1094.
To investigate the value of restriction display PCR (RD-PCR) as a novel and expedient sample labeling method for high-density 60-mer oligonucleotide microarray.
Peripheral blood samples from three volunteers were collected and the total RNA was extracted from the peripheral blood mononuclear cells and labeled with RD-PCR protocol, followed by hybridization with Agilent Human 1B oligonucleotide microarrays in a two-color comparison format. The RNA from the same subject was divided into two aliquot and labeled with Cy3 and Cy5 respectively. The spots with significant difference between the foreground and local background intensities and those without significant difference between Cy5 and Cy3 signal intensities were selected for analysis. SPSS software was used to perform the statistical tests and plot generation. VSN packages were used under R language to remove the systematic array and dye biases.
Totally 8744 common spots of the 3 microarrays were evaluated. The results demonstrated that RD-PCR could be a promising novel method for efficient labeling of microarray samples. Further analysis indicated the presence of adjustable biases derived from the array and incorporated dye in the labeling processes. The RD-PCR labeling showed better performance than the conventional approaches in regards to reproducibility of the quantitative signals for gene intensity and capability to label RNAs of lowly expressed genes.
Given the evidence of the feasibility of using RD-PCR labeling in the field of high-density long oligonucleotide microarray, further optimization of the protocol may unleash the full potential of this novel labeling method.
探讨限制性显示PCR（RD-PCR）作为一种新型便捷的样本标记方法用于高密度60聚体寡核苷酸微阵列的价值。
采集3名志愿者的外周血样本，从外周血单个核细胞中提取总RNA，采用RD-PCR方案进行标记，然后以双色比较模式与安捷伦人类1B寡核苷酸微阵列杂交。将同一受试者的RNA分成两份，分别用Cy3和Cy5标记。选择前景与局部背景强度有显著差异以及Cy5和Cy3信号强度无显著差异的斑点进行分析。使用SPSS软件进行统计检验和绘图。在R语言下使用VSN软件包去除系统阵列和染料偏差。
共评估了3个微阵列的8744个公共斑点。结果表明，RD-PCR可能是一种用于微阵列样本高效标记的有前景的新方法。进一步分析表明，在标记过程中存在来自阵列和掺入染料的可调节偏差。在基因强度定量信号的重现性以及标记低表达基因RNA的能力方面，RD-PCR标记比传统方法表现更好。
鉴于在高密度长寡核苷酸微阵列领域使用RD-PCR标记的可行性证据，进一步优化该方案可能释放这种新型标记方法的全部潜力。
