Real-time PCR expression profiling of microRNA

描述（由申请人提供）：microRNA是一类新发现的内源性小干扰RNA。MicroRNA与信使RNA结合并抑制蛋白质水平。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仍在很大程度上未知。已经提出了microRNA表达和癌发生之间的作用。目前缺乏灵敏的高通量方法来监测microRNA的表达。microRNA是具有挑战性的分子，因为microRNA前体由稳定的发夹组成，成熟的microRNA长度仅为22个核苷酸。我们建议评估敏感和特异性的实时PCR检测，以量化成熟和microRNA前体的表达。microRNA表达将在与人类癌症相关的许多重要生物学条件下进行分析。将在通过激光捕获显微切割分离的癌症和正常组织的特定切片中确定microRNA表达。将使用实时PCR和cDNA微阵列比较成熟和前体microRNA的表达。将在人胰腺癌的临床样品中研究microRNA表达。实时PCR应用于这类新的令人兴奋的调节RNA的无与伦比的灵敏度和特异性应该推动该领域进入新的方向，不仅在癌症，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.