Rapid quantitation of small RNA molecules

描述(申请人提供)：这项提案的总体目标是开发快速、高通量的方法来量化生物样本中小RNA分子的水平，如小干扰RNA(SiRNAs)和微小RNA(MiRNAs)。制药和生物技术公司开始测试siRNAs作为治疗剂。此外，miRNAs似乎是发育和疾病进展过程中基因表达的重要调节因子。不幸的是，生物医学研究人员缺乏快速、定量的方法来检测临床样本中的小RNA。这项拟议的研究将导致开发试剂盒和试剂，以快速检测和定量来自组织或细胞培养的样品中的siRNA或miRNA。将对三种不同的检测方法进行评估。第一种方法是一种新技术，它利用标记的蛋白质直接检测微板中的小RNA。第二种要检测的方法是杂交保护试验(HPA)。这种均相检测方法使用的是一种寡核苷酸探针，该探针内部标记有化学发光的吖啶酯基团。第三种方法是一种新的、基于专利的qRT-PCR方法。这种方法产生的聚合酶链式反应产物比miRNA模板大得多，从而可以通过标准的检测方法如TaqMan进行定量。第一阶段的具体目标：(1)建立一种快速、定量的平板检测方法，用于检测细胞培养和组织样本(200,000个细胞)中的miRNA和siRNA。(2)建立定量RT-PCR方法，用于检测细胞培养和组织样本(200,000个细胞)中的miRNA和siRNA。在第一阶段，这三种方法中的每一种都将使用纯合成miRNA开发，然后使用添加到复杂RNA混合物中的合成miRNA和siRNA进行优化。经过优化后，将通过测试它们对组织和细胞培养样本中siRNA和内源性miRNA进行量化的能力来直接比较这些方法。观察到的RNA水平将与使用核糖核酸酶保护试验测量的已知水平进行比较。然后，这三种方法将按照灵敏度、准确度、精密度、稳健性、速度、便利性和成本进行排序。在第二阶段，我们将把最有效的方法(S)应用到强大的、高通量的试剂盒中，以测量研究和临床样本中的siRNA和miRNA水平。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to develop rapid, high-throughput methods to quantify the levels of small RNA molecules such as small-interfering RNAs (siRNAs) and microRNAs (miRNAs) in biological samples. Pharmaceutical and biotechnology companies are beginning to test siRNAs as therapeutic agents. Also, miRNAs appear to be important regulators of gene expression during development and disease progression. Unfortunately biomedical researchers lack rapid, quantitative assays to detect small RNAs in clinical samples. The proposed research will result in the development of kits and reagents to rapidly detect and quantify siRNA or miRNA in samples derived from tissues or cell culture. Three different detection methods will be evaluated. The first method is a novel technique that utilizes a labeled protein to directly detect small RNAs in microplates. The second method to be tested is the hybridization protection assay (HPA). This homogeneous assay uses an oligonucleotide probe internally labeled with a chemiluminescent acridinium ester group. The third method is a novel, proprietary qRT-PCR based method. The PCR products generated by this method are much larger than the miRNA templates, enabling quantitation by standard detection methods such as TaqMan. Specific aims for Phase I: (1) Develop a rapid, quantitative plate-based assay for detecting miRNA and siRNA in cell culture and tissue samples (<200,000 cells). (2) Develop a quantitative RT-PCR procedure for measuring miRNA and siRNA in cell culture and tissue samples (<200,000 cells). In Phase I each of the three methods will be developed using pure synthetic miRNA and then optimized using synthetic miRNA and siRNA spiked into complex RNA mixtures. After optimization, the methods will be directly compared by testing their ability to quantify both siRNA and endogenous miRNA in samples from tissue and cell culture. Observed levels of the RNAs will be compared to known levels measured using the ribonuclease protection assay. The three methods will then be rank-ordered in terms of sensitivity, accuracy, precision, robustness, speed, convenience and cost. In Phase II, we will adapt the most effective method(s) into robust, high-throughput kits to measure siRNA and miRNA levels in research and clinical samples.