Rapid quantitation of small RNA molecules

小 RNA 分子的快速定量

• 批准号: 6833024
• 负责人: JOSEPH FRANCIS KREBS
• 金额: $ 19.65万
• 依托单位: AMBION, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-15 至 2006-05-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6833024
• 关键词: RNA; biotechnology; high throughput technology; nucleic acid probes; nucleic acid quantitation /detection; oligonucleotides; polymerase chain reaction; reagent /indicator; technology /technique development; tissue /cell culture

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.

描述（由申请人提供）：该提案的总体目标是开发快速，高通量的方法，以量化生物样品中小的RNA分子的水平，例如小互为互为RNA（siRNA）和microRNA（mirnas）。药物和生物技术公司开始将siRNA作为治疗剂进行测试。同样，miRNA似乎是发育和疾病进展过程中基因表达的重要调节剂。不幸的是，生物医学研究人员缺乏快速的定量测定，无法检测临床样本中的小RNA。拟议的研究将导致在源自组织或细胞培养的样品中快速检测和量化siRNA或miRNA的套件和试剂的发展。将评估三种不同的检测方法。第一种方法是一种新型技术，该技术利用标记的蛋白质直接检测微板岩中的小RNA。要测试的第二种方法是杂交保护测定法（HPA）。该均质测定法使用了用化学发光的递量酯组标记的寡核苷酸探针。第三种方法是一种新型的，专有的基于QRT-PCR的方法。该方法生成的PCR产物比miRNA模板大得多，可以通过标准检测方法（例如Taqman）进行定量。 I期的具体目的：（1）开发一种基于板块的快速，基于板块的测定法，用于检测细胞培养和组织样品中的miRNA和siRNA（<200,000个细胞）。 （2）开发一种定量的RT-PCR程序，用于测量细胞培养和组织样品中的miRNA和siRNA（<200,000个细胞）。在I期中，每种方法中的每种方法都将使用纯合成miRNA开发，然后使用合成miRNA和siRNA进行优化，并将siRNA尖刺到复杂的RNA混合物中。优化后，该方法将通过测试其在组织和细胞培养的样品中量化siRNA和内源miRNA的能力，直接比较。观察到的RNA水平将与使用核糖核酸酶保护测定法测量的已知水平进行比较。然后，这三种方法将以敏感性，准确性，精度，鲁棒性，速度，便利性和成本来排名。在第二阶段，我们将最有效的方法适应可靠的高通量试剂盒，以测量研究和临床样品中的siRNA和miRNA水平。