Development of a standardized kit for the isolation, enrichment, and high-sensitivity profiling of circular RNAs in the nervous system

开发用于神经系统中环状 RNA 的分离、富集和高灵敏度分析的标准化试剂盒

• 批准号: 8981779
• 负责人: Jarret Glasscock
• 金额: $ 91.87万
• 依托单位: COFACTOR GENOMICS, LLC
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8981779
• 关键词: Biological Assay; Biological Markers; Brain; Cell physiology; Client; Clinical; Computer software; Cytoplasm; Data; Development; Enzymes; Evaluation; Feedback; Fostering; Freezing; Health; Human; Human Biology; Laboratories; Lead; Libraries; Marketing; Measures; Mental disorders; Messenger RNA; Methods; MicroRNAs; Molecular Biology; Nervous system structure; Phase; Plasma; Play; Production; Protocols documentation; Quality Control; RNA; Reaction; Reporting; Research; Research Design; Research Personnel; Role; Sampling; Scientist; Sequence Homology; Services; Substance abuse problem; Temperature; Testing; Time; Tissues; Titrations; Transcript; Translating; Untranslated RNA; biobank; circular RNA; clinically relevant; cofactor; design; human disease; interest; nervous system disorder; neuron development; public health relevance; qubit; research and development; success; user-friendly; volunteer

 DESCRIPTION (provided by applicant): Long-term objectives: Modified ribonucleic acids (RNA) are important regulators of numerous cellular processes, and are increasingly being associated with human diseases. For example, non-coding microRNAs play significant roles in neuronal development, and can serve as biomarkers for psychiatric disorders. Another such modified RNA is circular RNA (circRNA), a non-coding transcript present in the cytoplasm. Since circRNAs are present in lower abundance than other RNA molecules and share sequence homology with mRNA, they are difficult to isolate from total RNA. Our long-term objective is to further develop and distribute a molecular biology kit, which will foster broader research in this nascent field and expand our understanding of the role of circular RNAs in neuronal development, substance abuse, and psychiatric disorders. We have established four Phase II Aims: (1) Method optimization, (2) Clinical Sample Applicability + Optimization, (3) Internal Kit Testing/Cofactor circRNA Service, and (4) External Field Testing/ Kit Beta Testers. Research Design: Aim 1: We will utilize universal human brain reference RNA containing a known quantity of circRNA control, and our optimized enzyme-mix to determine ideal reaction parameters (i.e. primer design, temperature, and incubation time) that will lead to the most robust enrichment of ciRNA species. Input titrations will determine the threshold and range of total RNA input for the optimized protocol. Success will be measured by qPCR and sequencing metrics on libraries passing our quality control measures. Aim 2: We will obtain commercially available biobank samples such as FFPE tissue, frozen brain, and plasma to test the kit applicability and robustness on clinically relevant samples. Quality metrics assayed by Qubit, Bioanalyzer/Tapestation, and Nanodrop will be recorded for the corresponding sequencing libraries to identify kit robustness. In addition, samples provided by commercial and academic partners will further bolster our metric reports for varying sample types and input amounts. Aim 3: Our production scientists will translate the R&D SOP into a production service offering with client-submitted samples. We will continue to collect quality metrics on these samples in the production setting to determine expected yields for library construction and circRNA control enrichment. We will use the data generated from both our R&D and production setting to develop and optimize our analysis software, ActiveSite, to incorporate circRNA enrichment reports. Aim 4: We have interested commercial and academic partners that have volunteered to test our circRNA kit in their respective laboratories. Our partners will collect quality metric scoes about the samples, generate circRNA- enriched samples, and provide these to us for evaluation and sequencing. We will utilize feedback from these Beta testers to refine the SOP and our market strategy.

 描述(由申请人提供)：长期目标：修饰的核糖核酸(RNA)是许多细胞过程的重要调节者，并且越来越多地与人类疾病有关。例如，非编码的microRNA在神经元发育中发挥着重要作用，并可以作为精神疾病的生物标记物。另一种这种修饰的RNA是环状RNA(CircRNA)，它是一种存在于细胞质中的非编码转录本。由于CircRNAs的丰度低于其他RNA分子，并且与mRNA具有序列同源性，因此很难从总RNA中分离出它们。我们的长期目标是进一步开发和分发分子生物学试剂盒，这将促进这一新兴领域的更广泛研究，并扩大我们对环状RNA在神经元发育、物质滥用和精神障碍中的作用的理解。我们建立了四个第二阶段的目标：(1)方法优化，(2)临床样本适用性+优化，(3)内部试剂盒测试/辅因子CircRNA服务，以及(4)外部现场测试/试剂盒Beta测试器。研究设计：目标1：我们将利用包含已知数量的CircRNA控制的通用人脑参考RNA和我们优化的酶组合来确定理想的反应参数(即引物设计、温度和孵化时间)，以导致最稳健的ciRNA种类的丰富。输入滴定将确定优化方案的总RNA输入阈值和范围。成功的衡量标准是通过我们的质量控制措施对图书馆进行定量聚合酶链式反应和测序。目的2：我们将获得商业上可用的生物库样本，如FFPE组织、冰冻脑和血浆，以测试试剂盒在临床相关样本上的适用性和稳健性。将为相应的测序库记录由Qubit、BioAnalyzer/Tapestation和NanoDrop分析的质量指标，以确定试剂盒的稳健性。此外，商业和学术合作伙伴提供的样本将进一步支持我们针对不同样本类型和投入数量的指标报告。目标3：我们的生产科学家将把研发SOP转化为客户提交的样品的生产服务产品。我们将继续在生产环境中收集这些样本的质量指标，以确定文库建设和CircRNA控制浓缩的预期产量。我们将使用从我们的研发和生产环境中产生的数据来开发和优化我们的分析软件ActiveSite，以纳入CircRNA浓缩报告。目标4：我们有感兴趣的商业和学术合作伙伴，他们自愿在各自的实验室测试我们的CircRNA试剂盒。我们的合作伙伴将收集有关样本的质量度量分数，生成富含CircRNA的样本，并将其提供给我们进行评估和测序。我们将利用这些Beta测试员的反馈来完善SOP和我们的市场战略。