Real-time PCR expression profiling of microRNA

描述（申请人提供）：microRNA是一类新发现的内源性小干扰RNA。MicroRNA结合信使RNA并翻译抑制蛋白水平。虽然仅在人类中就发现了300多种microrna，但它们的生物学功能、靶点、表达水平和在疾病中的作用在很大程度上仍然未知。microRNA表达与癌变之间的作用已被提出。目前缺乏敏感的、高通量的方法来监测microrna的表达。microRNA是具有挑战性的分子，因为microRNA前体是一个稳定的发夹，成熟的microRNA只有22个核苷酸的长度。我们建议评估敏感性和特异性实时PCR分析，以量化成熟和microRNA前体的表达。microRNA的表达将在许多与人类癌症有关的重要生物学条件下进行分析。microRNA的表达将在通过激光捕获显微解剖分离的癌症和正常组织的特定切片中确定。成熟和前体microrna的表达将通过实时PCR和cDNA微阵列进行比较。将在人类胰腺癌的临床样本中研究microRNA的表达。实时PCR无与伦比的灵敏度和特异性应用于这类令人兴奋的新型调控rna，将推动该领域进入新的方向，不仅在癌症领域，而且在人类健康的其他领域。

DESCRIPTION (provided by applicant): microRNA is a newly discovered class of endogenous, small interfering RNA. MicroRNA binds to messenger RNA and translationally represses protein levels. While over 300 microRNAs have been discovered in humans alone, their biological function, targets, expression levels and role in disease remain largely unknown. A role between microRNA expression and carcinogenesis has been proposed. There is a lack of sensitive, high-throughput methodologies to monitor the expression of microRNAs. microRNA are challenging molecules to quantify because the microRNA precursors consists as a stable hairpin and the mature microRNA is only 22 nucleotides in length. We propose to evaluate sensitive and specific real-time PCR assays to quantify the expression of the mature and microRNA precursors. The microRNA expression will be analyzed in a number of important biological conditions relating to human cancer. The microRNA expression will be determined in specific sections of cancer and normal tissue isolated by laser-capture microdissection. The expression of mature and precursor microRNAs will be compared to using real-time PCR and a cDNA micro array. microRNA expression will be studied in clinical samples of human pancreatic cancer. The unparallel sensitivity and specificity of real-time PCR as applied to this new and exciting class of regulatory RNAs should propel the field into new directions not only in cancer but in other areas of human health.