Castoldi Mirco, Schmidt Sabine, Benes Vladimir, Hentze Matthias W, Muckenthaler Martina U
Molecular Medicine Partnership Unit (MMPU), Im Neuenheimer Feld 153, Heidelberg 69120, Germany.
Nat Protoc. 2008;3(2):321-9. doi: 10.1038/nprot.2008.4.
MicroRNAs (miRNAs) represent a class of short (22 nt) noncoding RNAs that control gene expression post-transcriptionally. Microarray technology is frequently applied to monitor miRNA expression levels but is challenged by (i) the short length of miRNAs that offers little sequence for appending detection molecules; (ii) low copy number of some miRNA; and (iii) a wide range of predicted melting temperatures (Tm) versus their DNA complementary sequences. We recently developed a microarray platform for genome-wide profiling of miRNAs (miChip) by applying locked nucleic acid (LNA)-modified capture probes. Here, we provide detailed protocols for the generation of the miChip microarray platform, the preparation and fluorescent labeling of small RNA containing total RNA, its hybridization to the immobilized LNA-modified capture probes and the post-hybridization handling of the microarray. Starting from the intact tissue sample, the entire protocol takes approximately 3 d to yield highly accurate and sensitive data about miRNA expression levels.
微小RNA（miRNA）是一类短（22个核苷酸）的非编码RNA，可在转录后水平控制基因表达。微阵列技术经常用于监测miRNA的表达水平，但面临以下挑战：（i）miRNA长度短，可供连接检测分子的序列很少；（ii）一些miRNA的拷贝数低；（iii）与其DNA互补序列相比，预测的解链温度（Tm）范围很广。我们最近通过应用锁核酸（LNA）修饰的捕获探针开发了一个用于miRNA全基因组分析的微阵列平台（miChip）。在此，我们提供了生成miChip微阵列平台、制备含小RNA的总RNA并进行荧光标记、将其与固定化的LNA修饰捕获探针杂交以及微阵列杂交后处理的详细方案。从完整的组织样本开始，整个方案大约需要3天时间才能产生关于miRNA表达水平的高度准确和敏感的数据。
