Massively parallel quantitative single-cell PCR with qCell Arrays

 描述(由申请人提供)：使命生物公司开发的仪器将通过快速和准确地执行单细胞核酸分析来加速与人类健康相关的细胞异质性的研究。我们之前介绍了一种新的分选技术--聚合酶激活细胞分选(PACS)，它不仅具有高通量，而且能够以无与伦比的灵敏度和特异度分析各种核酸生物标志物。使用在微流控液滴中执行的多重TaqMan聚合酶链式反应分析，该系统能够询问单个细胞转录本、基因组DNA、突变或非编码RNA的特定组合的表达，这些靶标不适合基于抗体的检测。虽然该平台已被证明广泛用于识别和分选许多稀有和独特的细胞，但它依赖于终点PCR反应，无法准确区分仅在核酸拷贝数上存在微小差异的相似细胞类型。这项SBIR建议的目标是开发一个对数万个细胞进行精确的单细胞核酸定量的平台。真正具有单细胞分辨率的定量能力要求在每个PCR周期结束时测量和记录TaqMan反应荧光。要做到这一点，我们的单细胞液滴反应，我们必须建立一个微流控芯片，qCell阵列，可以允许热循环，荧光成像和空间跟踪所有液滴。这种方法提供的能力将代表着对目前的定量单细胞定量定量聚合酶链式反应方法的突破，这些方法昂贵、劳动密集型和/或缺乏分析大量细胞所需的吞吐量。QCell阵列方法的智力优势来自于其核心能力，即为大规模并行、定量的单细胞遗传和转录分析提供快速和低成本的解决方案，以分析大量不同种类的细胞。细胞异质性对生物功能和疾病的影响是人类免疫学、干细胞生物学、传染病和癌症研究中至关重要的问题。通过分析混合群体中的单个细胞，有可能识别对人类健康和发育至关重要的稀有细胞群体或瞬时细胞状态，否则无法通过整体测量观察到这些状态。QCell阵列平台的商业化将使对这些异质细胞群体中的基因和转录本进行更详细和全面的分析成为可能。QCell阵列依靠成熟的分子生物学方法、先进的微流控技术和坚实的知识产权基础。第一阶段的资金将导致开发所需的、在技术上可实现的研究和诊断工具，在生物医学市场具有很高的影响潜力。

 DESCRIPTION (provided by applicant): Mission Bio, Inc. develops instrumentation that will accelerate the investigation of cellular heterogeneity relevant to human health by rapidly and accurately performing single-cell nucleic acid analysis. We previously introduced a new sorting technology, PCR-activated cell sorting (PACS), which is both high in throughput and able to analyze a wide variety of nucleic acid biomarkers with unmatched sensitivity and specificity. Using multiplexed TaqMan PCR assays performed in microfluidic droplets, the system is able to interrogate individual cells for the expression of specific combinations of transcripts, genomic DNA, mutations or non-coding RNAs, targets not amenable to antibody-based detection. While this platform has proven widely useful for identifying and sorting many rare and unique cells, it relies on endpoint PCR reactions and is not able to precisely discriminate between similar cell types possessing only minor differences in nucleic acid copy number. The objective of this SBIR proposal is to develop a platform for performing precise single-cell nucleic acid quantitation on tens of thousands of cells. True quantitative capability with single-cell resolution requires that the TaqMan reaction fluorescence be measured and recorded at the end of each cycle of PCR. To do this with our single-cell droplet reactions, we must build a microfluidic chip, the qCell Array, that can permit thermocycling, fluorescence imaging and spatial tracking of all droplets. The capability provided by such an approach would represent a breakthrough over current methods for quantitative single-cell qPCR that are expensive, labor intensive and/or lack the throughput necessary to analyze large numbers of cells. The intellectual merits of the qCell Array approach stem from the core ability to deliver a rapid and low- cost solution for massively parallel, quantitative single-cell genetic and transcriptional analysis on large heterogeneous populations of cells. The impact of cellular heterogeneity on biological function and disease is crucially important to questions in human immunology, stem cell biology, infectious disease and cancer research. By analyzing individual cells within a mixed population, it is possible to identif rare cell populations or transient cell states critical to human health and development that are otherwise unobservable by ensemble measurements. Commercialization of the qCell Array platform will enable a more detailed and comprehensive analysis of the genes and transcripts in these heterogeneous cell populations. The qCell Array relies on proven molecular biology approaches, advanced microfluidics, and a solid foundation of intellectual property. Phase I funding will result in the development of a needed and technically achievable research and diagnostic tool with high impact potential in the biomedical marketplace.