Efficient labeling of microRNA for microarray analysis

描述（由申请人提供）：6。MicroRNA（miRNAs）是一类长度约为19-23个核苷酸的非编码小RNA，通过与特定靶mRNA的3'非翻译区序列结合来调节基因表达;单个miRNAs可能与多达200个不同的靶点结合并调节基因表达。直到最近才发现，并且被认为是细胞增殖和分化所必需的，很明显，miRNA活性的异常有助于人类疾病（包括癌症）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（其提供很少的序列用于设计微阵列捕获序列或附着标签）以及它们仅占总RNA样品质量的约0.01%的事实，这使得分析miRNA表达水平变得困难。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。标记IT（r）试剂（Mirus Bio）利用烷基化化学将可检测的标记物如Cy 3和Cy 5直接共价连接到核酸。使用Label IT试剂标记miRNA样本可生成微阵列表达谱，可一致识别酶标记方法无法检测到的miRNA，包括各种癌症中记录的潜在诊断标记物。用于诊断、治疗和预防人类疾病的miRNA表达谱分析是一个快速扩展的研究领域，其关键取决于样品中存在的所有miRNA种类的成功鉴定。为了确定微阵列分析中使用Label IT技术和酶标记方法生成的miRNA表达谱的差异数量，将使用化学和酶方法标记来自人肿瘤/正常细胞系对的miRNA富集样品，与至少两种不同商业微阵列的多个重复样品杂交，并通过独立方法验证表达谱的差异。优化临床相关样本的标签IT标签（其中miRNA的量可能是有限的），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，并将包括确定非-扩增的材料需要产生代表性的微阵列表达谱和通用标签IT标记处理协议的发展临床RNA样品。此外，标签IT技术将优化用于小鼠和人类癌症标本的微阵列miRNA表达谱。我们预计，与目前可用的酶标记方法相比，临床研究和诊断样本中的miRNA的化学标记将为准确的表达谱分析提供许多关键优势。7. MicroRNAs（miRNAs）是一组通常调节细胞生长的小分子，但当它们的表达改变时，它们可能会导致癌症等疾病。微阵列是一种用于观察给定样品中所有基因的总表达的方法，并且这些miRNA在患病组织中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）试剂通过微阵列化学标记miRNA进行检查，与其他标记技术相比，其表达谱更准确和完整，在临床研究和人类疾病诊断方面具有明显优势。

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.