Efficient Labeling of MicroRNA for Microarray Analysis

描述（由申请人提供）： 最近发现了一类新的内源性小RNA（称为microRNA或miRNA），其介导哺乳动物细胞中的天然RNA干扰现象。特定的miRNA在不同的细胞类型、发育阶段和疾病状态（包括某些癌症）中表达。对miRNA表达模式的研究需要开发新的分析方法和工具，因为小尺寸的miRNA（约22个核苷酸）不容易适用于现有技术。微阵列是研究miRNA表达模式的理想高通量方法，将来可能用于miRNA诊断。高通量表达谱分析方法如微阵列需要用荧光染料有效地标记miRNA样品。理想的miRNA标记方法将等同地标记小RNA并且没有序列偏差，不会改变标记的miRNA的解链温度，将具有灵敏的检测限，并且将需要最小的样品操作或改变。目前没有一种标记方法符合所有这些标准。我们建议开发一种高效、简单的RNA标记技术，该技术将保留原始RNA样品，向每个RNA靶标添加单个标记，在标记中不显示序列偏差，对杂交或解链温度的影响可以忽略不计，并且优先标记含有复杂RNA混合物的样品中的小RNA分子，如miRNA。我们将展示这种直接标记方法的原型短寡miRNA微阵列上的性能。

DESCRIPTION (provided by applicant): A novel class of endogenous small RNAs (termed microRNAs, or miRNAs) has recently been discovered that mediates native RNA interference phenomena in mammalian cells. Specific miRNAs are expressed in different cell types, stages of development, and disease states, including certain cancers. Research on miRNA expression patterns requires the development of new analytical methods and tools because the small size of miRNAs (approximately 22 nucleotides) is not readily amenable to existing technologies. Microarrays represent an ideal high throughput method for research on miRNA expression patterns and, in the future, possibly miRNA diagnostics. High throughput expression profiling methods such as microarrays require miRNA samples to be efficiently labeled with a fluorescent dye. The ideal miRNA labeling method would label small RNAs equivalently and without sequence bias, would not alter the melting temperature of the labeled miRNA, would have a sensitive limit of detection, and would require minimal sample manipulation or alteration. No current labeling methods meet all of these criteria. We propose to develop a highly efficient, straightforward RNA labeling technology that would preserve the original RNA sample, add a single label to each RNA target, exhibit no sequence bias in labeling, have a negligible effect on hybridization or melting temperature, and preferentially label small RNA molecules such as miRNA in samples containing complex mixtures of RNA. We will demonstrate the performance of this direct labeling method on a prototype short-oligo miRNA microarray.