Castoldi Mirco, Benes Vladimir, Hentze Matthias W, Muckenthaler Martina U
Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg, Im Neuenheimer Feld 156, D-69210 Heidelberg, Germany.
Methods. 2007 Oct;43(2):146-52. doi: 10.1016/j.ymeth.2007.04.009.
As key regulators of post-transcriptional gene expression, it is important to monitor the expression of microRNAs (miRNA) in diverse physiological and pathophysiological processes. Here, we describe a method for sensitive and accurate microarray-based expression profiling of miRNAs. The protocol focuses on the use of locked nucleic acid (LNA)-modified capture probes. LNAs are bicyclic nucleotide analogues that significantly increase the melting temperature (T(m)) of hybrids with miRNAs. Mixed LNA/DNA capture probes thus can be designed for equal T(m)s for all miRNAs, which naturally cover a range between 45 and 74 degrees C. The protocols established are easy to apply, as they do not require RNA size selection and/or amplification of miRNAs. Moreover, they enable high affinity hybridizations yielding accurate signals that discriminate between single nucleotide differences and hence closely related miRNA family members.
作为转录后基因表达的关键调节因子，监测微小RNA（miRNA）在各种生理和病理生理过程中的表达非常重要。在此，我们描述了一种基于微阵列的灵敏且准确的miRNA表达谱分析方法。该方案重点在于使用锁核酸（LNA）修饰的捕获探针。LNA是双环核苷酸类似物，可显著提高与miRNA杂交体的解链温度（T(m)）。因此，可以设计混合的LNA/DNA捕获探针，使所有miRNA的T(m)相等，其天然范围在45至74摄氏度之间。所建立的方案易于应用，因为它们不需要对miRNA进行RNA大小选择和/或扩增。此外，它们能够实现高亲和力杂交，产生准确的信号，可区分单核苷酸差异，从而区分密切相关的miRNA家族成员。
