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microRNAs，或miRNAs)，它介导了哺乳动物细胞中的天然RNA干扰现象。特定的miRNAs在不同的细胞类型、发育阶段和疾病状态中表达，包括某些癌症。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miRNAs(约22个核苷酸)不容易适应现有技术。微阵列是研究miRNA表达模式和未来可能的miRNA诊断的理想的高通量方法。高通量表达谱方法，如微阵列，需要用荧光染料有效地标记miRNA样本。理想的miRNA标记方法将等量地标记小RNA，没有序列偏差，不会改变标记的miRNA的熔融温度，具有灵敏的检测限，并且需要最少的样品操作或改变。目前没有一种标签方法满足所有这些标准。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样本，为每个RNA靶标添加一个单一标记，标记时不表现出序列偏差，对杂交或熔融温度的影响可以忽略不计，并优先标记包含复杂RNA混合物的样本中的小RNA分子，如miRNA。我们将在一个原型的短寡核苷酸miRNA微阵列上演示这种直接标记方法的性能。

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.