Yang Yunfeng
Biosciences Division, Oak Ridge National Laboratory, TN, Oak Ridge, USA.
Methods Mol Biol. 2009;544:439-50. doi: 10.1007/978-1-59745-483-4_29.
DNA microarray has become a mainstream technology to explore gene expression profiles, identify novel genes involved in a biological process of interest and predict their function, and determine biomarkers that are relevant to a given phenotype or disease. Typical two-channel microarray studies use an experimental design called the complementary DNA (cDNA) reference method, in which samples from test and control conditions are compared directly on a microarray slide. A substantial limitation of this strategy is that it is nearly impossible to compare data between experiments because the reference sample composition is subjected to changes at the level of experimental design and thereby not consistent from one experiment to another. Using genomic DNA as common reference will effectively overcome this limitation. This chapter describes detailed methods to prepare genomic DNA of high quality, label with fluorescent dye, co-hybridize with cDNA samples, and the subsequent data analyses. In addition, notes are provided to help the readers to obtain optimal results using the procedure.
DNA微阵列已成为一种主流技术,用于探索基因表达谱、识别参与感兴趣的生物学过程的新基因并预测其功能,以及确定与给定表型或疾病相关的生物标志物。典型的双通道微阵列研究使用一种称为互补DNA(cDNA)参照法的实验设计,其中来自测试和对照条件的样本在微阵列载玻片上直接进行比较。该策略的一个重大局限性在于,几乎不可能在不同实验之间比较数据,因为参照样本的组成在实验设计层面会发生变化,从而在不同实验之间不一致。使用基因组DNA作为通用参照将有效克服这一局限性。本章详细描述了制备高质量基因组DNA、用荧光染料标记、与cDNA样本共杂交以及后续数据分析的方法。此外,还提供了注释,以帮助读者使用该程序获得最佳结果。