Efficient labeling of microRNA for microarray analysis

描述（由申请人提供）：MicroRNAs （miRNAs）是一组小的非编码rna，长度约为19-23个核苷酸，通过结合特定靶mrna的3'非翻译区序列来调节基因表达；单个miRNA可能结合并调节多达200个不同的靶标。直到最近才被发现，并被认为是细胞增殖和分化的必要条件，很明显，miRNA活性的异常有助于包括癌症在内的人类疾病的发病机制。微阵列是高通量基因表达分析的理想选择；然而，当分析miRNA表达水平时，由于其固有的小尺寸（这为设计微阵列捕获序列或附加标签提供了很少的序列）以及它们仅占总RNA样品质量的~0.01%，这使得分析变得困难。通常，对于微阵列应用，使用基于聚(a)聚合酶的方法将荧光染料如Cy(tm)3和Cy(tm)5添加到mirna中。Label IT(r)试剂（Mirus Bio）利用烷基化化学将可检测的标记物（如Cy3和Cy5）直接与核酸共价连接。用Label IT试剂标记miRNA样品产生的微阵列表达谱一致地识别酶标记方法无法检测到的miRNA，包括在各种癌症中记录的潜在诊断标记。用于诊断、治疗和预防人类疾病的miRNA表达谱是一个迅速发展的研究领域，它严重依赖于样品中存在的所有miRNA物种的成功鉴定。为了确定在微阵列分析中使用Label IT技术和酶标记方法生成的miRNA表达谱中的差异数量，将使用化学和酶方法标记来自人类肿瘤/正常细胞系对的富含miRNA的样品，将其杂交到至少两种不同的商业微阵列的多个重复中，并通过独立的方法验证表达谱的差异。为了优化临床相关样品的Label IT标记（其中miRNA的数量可能有限），将使用一个肿瘤/正常细胞系对进行各种miRNA富集和扩增程序的系统检查，并将包括确定生成具有代表性的微阵列表达谱所需的最小非扩增材料量，以及开发用于处理临床RNA样品的通用Label IT标记方案。此外，Label IT技术将被优化用于小鼠和人类癌症标本的微阵列miRNA表达谱分析。我们预计，与目前可用的酶标记方法相比，来自临床研究和诊断标本的miRNA化学标记将为准确的表达谱提供许多关键优势。7. MicroRNAs （miRNAs）是一组通常调节细胞生长的小分子，但当它们的表达发生改变时，可能会导致癌症等疾病。微阵列是一种观察给定样本中所有基因总表达的方法，这些病变组织中mirna的表达谱可用于诊断和建议治疗方案。与其他使用酶的方法相比，Label IT(r)试剂对mirna进行化学标记，以供微阵列检测，与其他标记技术相比，其表达谱更准确、更完整，对人类疾病的临床研究和诊断具有明显的优势。

DESCRIPTION (provided by applicant): 6. Project Summary/Abstract MicroRNAs (miRNAs) are a group of small, non-coding RNAs, approximately 19-23 nucleotides in length, that regulate gene expression by binding to sequences within the 3' untranslated regions of specific target mRNAs; a single miRNA might bind to and regulate as many as 200 diverse targets. Only recently discovered, and thought to be essential for cell proliferation and differentiation, it is clear that abnormalities in miRNA activity contribute to the pathogenesis of human diseases including cancer. Microarrays are ideal for high-throughput analysis of gene expression; however, when analyzing miRNA expression levels this is made difficult by their inherent small size (which provides little sequence for designing microarray capture sequences or attaching label) and the fact that they represent only ~0.01% of the mass of a total RNA sample. Typically, for microarray applications, fluorescent dyes such as Cy(tm)3 and Cy(tm)5 are added to miRNAs using a poly(A) polymerase-based method. Label IT(r) reagents (Mirus Bio) utilize alkylating chemistry to covalently link a detectable marker such as Cy3 and Cy5 directly to nucleic acids. Labeling miRNA samples with the Label IT reagents generates microarray expression profiles that consistently identify miRNAs that the enzymatic labeling methods fail to detect, including potential diagnostic markers documented in various cancers. Expression profiling of miRNAs for the diagnosis, treatment, and prevention of human diseases is a rapidly expanding field of research that is critically dependent on the successful identification of all miRNA species present in the sample. To determine the number of discrepancies in the miRNA expression profiles generated using Label IT technology and enzymatic labeling methodologies in microarray analyses, miRNA-enriched samples from human tumor/normal cell line pairs will be labeled using chemical and enzymatic methods, hybridized to multiple replicates of at least two different commercial microarrays, and the differences in expression profiles validated by independent methods. To optimize Label IT labeling for clinically relevant samples (where the amount of miRNA may be limiting), a systematic examination of various miRNA enrichment and amplification procedures will be performed using one tumor/normal cell line pair, and will include a determination of the minimum amount of non-amplified material needed to generate representative microarray expression profiles and the development of universal Label IT labeling protocols for processing clinical RNA samples. Furthermore, the Label IT technology will be optimized for microarray miRNA expression profiling of mouse and human cancer specimens. We anticipate that chemical labeling of miRNA from clinical research and diagnostic specimens will offer many critical advantages for accurate expression profiling compared to currently available enzymatic labeling methods. 7. Project Narrative MicroRNAs (miRNAs) are a group of small molecules that normally regulate cell growth, but they may cause diseases such as cancer when their expression is altered. Microarrays are a method for looking at the total expression of all genes in a given sample, and these expression profiles of miRNAs in diseased tissues may be used for diagnosis and to suggest treatment options. In contrast to other methods that use enzymes, Label IT(r) reagents chemically label miRNAs for examination by microarrays, resulting in a more accurate and complete expression profile compared to other labeling technologies with clear advantages for clinical research and diagnosis of human diseases.