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

基本信息

  • 批准号:
    9087198
  • 负责人:
  • 金额:
    $ 49.09万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

 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剂,并且与人类疾病的关系日益密切。例如,非编码 microRNA 在神经元发育中发挥重要作用,并且可以作为精神疾病的生物标志物。另一种此类修饰的 RNA 是环状 RNA (circRNA),一种存在于细胞质中的非编码转录物。由于 circRNA 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、药物滥用和精神疾病中作用的理解。我们制定了四个 II 期目标:(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 测试仪。研究设计:目标 1:我们将利用含有已知数量的 circRNA 对照的通用人脑参考 RNA 和我们优化的酶混合物来确定理想的反应参数(即引物设计、温度和孵育时间),从而实现 ciRNA 物种的最稳健富集。输入滴定将确定优化方案的总 RNA 输入的阈值和范围。成功与否将通过 qPCR 和通过我们质量控制措施的文库的测序指标来衡量。目标 2:我们将获取市售生物样本库样本,例如 FFPE 组织、冷冻大脑和血浆,以测试试剂盒对临床相关样本的适用性和稳健性。将记录相应测序文库由 Qubit、Bioanalyzer/Tapestation 和 Nanodrop 检测的质量指标,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输入量的指标报告。目标 3:我们的生产科学家将研发 SOP 转化为生产服务,并提供客户提交的样品。我们将继续在生产环境中收集这些样本的质量指标,以确定文库构建和 circRNA 控制富集的预期产量。我们将使用研发和生产环境生成的数据来开发和优化我们的分析软件 ActiveSite,以纳入 circRNA 富集报告。目标 4:我们有感兴趣的商业和学术合作伙伴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样本的质量指标,生成富含 circRNA 的样本,并将这些样本提供给我们进行评估和测序。我们将利用这些 Beta 测试人员的反馈来完善 SOP 和我们的市场策略。

项目成果

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Jarret Glasscock其他文献

Jarret Glasscock的其他文献

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{{ truncateString('Jarret Glasscock', 18)}}的其他基金

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
  • 财政年份:
    2014
  • 资助金额:
    $ 49.09万
  • 项目类别:
Developing a standardized kit and normal reference profile for circular RNAs in the nervous system
开发神经系统中环状 RNA 的标准化试剂盒和正常参考谱
  • 批准号:
    8841991
  • 财政年份:
    2014
  • 资助金额:
    $ 49.09万
  • 项目类别:

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