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与信使RNA结合，并在翻译上抑制蛋白质水平。虽然仅在人类身上就发现了300多个microRNAs，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不清楚。已经提出了在microRNA表达和肿瘤发生之间的作用。目前还缺乏灵敏、高通量的方法来监测microRNAs的表达。MicroRNA是具有挑战性的定量分子，因为microRNA前体是一个稳定的发夹，而成熟的microRNA只有22个核苷酸的长度。我们建议评估灵敏和特异的实时聚合酶链式反应方法，以量化成熟和微小RNA前体的表达。将在一些与人类癌症有关的重要生物学条件下分析microRNA的表达。微小RNA的表达将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来确定。成熟的和前体的microRNAs的表达将与使用实时荧光聚合酶链式反应和cDNA微阵列进行比较。将研究人胰腺癌临床样本中microRNA的表达。实时聚合酶链式反应的无与伦比的敏感性和特异性应用于这类新的和令人兴奋的调控RNA，不仅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.