Schinke-Braun Martina, Couget Jennifer A
Novartis Institutes for BioMedical Research, Cambridge, MA, USA.
Methods Mol Biol. 2007;366:13-40. doi: 10.1007/978-1-59745-030-0_2.
Large-scale microarray expression profiling studies have helped us to understand basic biological processes and to classify and predict the prognosis of cancers; they have also accelerated the identification of new drug targets. Affymetrix GeneChip probe arrays are high-density oligonucleotide microarrays that are available for many prokaryotic and eukaryotic species. Affymetrix human and mouse whole-genome microarrays analyze the expression level of up to 47,000 transcripts and variants. Each transcript is measured by 11 probe pairs, which consist of a perfect match 25mer oligonucleotide (PM) and a 25mer mismatch oligonucleotide (MM) that contains a single base pair mismatch in the central position. The PM/MM design is used for identification and subtraction of nonspecific hybridization and background signals. Advantages of Affymetrix GeneChip arrays include highly standardized array fabrication, as well as standardized target preparation, hybridization, and processing protocols, resulting in low technical variability and good reproducibility between experiments. This chapter describes the standard assay procedures for isolating total RNA from heart tissue, generating a biotin-labeled target for expression analysis, processing of Affymetrix GeneChip probe arrays using the Affymetrix instrument system, and quality control measures.
大规模微阵列表达谱研究有助于我们理解基本生物学过程，对癌症进行分类和预测预后；它们还加速了新药物靶点的识别。Affymetrix基因芯片探针阵列是高密度寡核苷酸微阵列，可用于许多原核和真核生物物种。Affymetrix人类和小鼠全基因组微阵列可分析多达47,000个转录本和变体的表达水平。每个转录本由11对探针测量，这些探针由一个完全匹配的25聚体寡核苷酸（PM）和一个在中心位置含有一个碱基对错配的25聚体错配寡核苷酸（MM）组成。PM/MM设计用于识别和扣除非特异性杂交和背景信号。Affymetrix基因芯片阵列的优点包括高度标准化的阵列制造，以及标准化的靶标制备、杂交和处理方案，从而导致实验间技术变异性低且重现性好。本章描述了从心脏组织中分离总RNA、生成用于表达分析的生物素标记靶标、使用Affymetrix仪器系统处理Affymetrix基因芯片探针阵列以及质量控制措施的标准检测程序。
