In Situ Single Cell Laser Lysis and Downstream qRT-PCR Profiling

原位单细胞激光裂解和下游 qRT-PCR 分析

• 批准号: 8414019
• 负责人: Deirdre R. Meldrum
• 金额: $ 21.86万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-26 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8414019
• 关键词: Affect; Arizona; Automation; Barrett Esophagus; Biocompatible Materials; Biological; Biomedical Research; Biophysics; Biopsy; Cells; Cellular Stress; Cellular biology; Clinic; Clinical; Cytolysis; Development; Devices; Emulsions; Energy Transfer; Engineering; Esophageal; Event; Fresh Tissue; Future; Gene Expression; Genes; Germ Cells; Goals; Grant; Heat-Shock Proteins 70; Heterogeneity; Hour; Human; In Situ; Individual; Lasers; Life; Light; Location; Malignant Neoplasms; Messenger RNA; Methods; Microfluidics; Microscope; Molecular Biology; Morphology; Oils; One-Step dentin bonding system; Photons; Physiologic pulse; Population; Population Heterogeneity; Process; Protocols documentation; Reaction; Reagent; Research; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Signal Transduction; Slice; Solutions; Source; Specimen; Structure; System; Techniques; Technology; Testing; Time; Tissues; base; biosignature; clinical application; innovation; interest; intestinal crypt; mRNA Expression; microsystems; nanometer; nanoscale; neoplastic cell; novel diagnostics; prevent; response; sample collection; single cell analysis; time interval; tool; tumor; two-photon

DESCRIPTION (provided by applicant): Current RT-PCR analysis of single-cells has been strictly limited to analysis of disassociated cells, preventing its compatibility with in-situ analsis of cell states and loses information of initial cell location and morphology within the native tissue. We propose to develop an innovative microfluidic based tool for clinical and scientific users to analyze gene expression heterogeneity, in situ, using single-cell mRNA expression analysis. The device uses a two-photon laser to serially lyse individual cells at known coordinates within a 3D tissue. Differing from conventional single-photon laser lysis, two-photon laser lysis relies on the nonlinear interaction between an ultrafast pulsed light source and the biological material to achieve an energy transfer to the cell precisely within the nanometer-scale focal volume. The lysate is immediately transported to an emulsion-based (oil-droplet) qRT-PCR module to profile mRNA expression. Carryover contamination between sequentially lysed cells is minimized by optimizing laser power and by using hydrodynamic flow focusing with precise flow rate control. Because of the small scale of the microfluidic channels, the total volume flux for sample processing is reduced to microliters, the elapsed time interval between cell lysing and lysate encapsulation is on the order of seconds, and completion of qRT-PCR is on the order of one hour. This technology is well suited to basic biomedical research and clinical applications such as assessing tumor cell population heterogeneity in single-cell gene expression. Additionally, the technology is also amenable to future developments to increase the number of genes that can be quantified. The ultimate implementation would be a highly multiplexed platform capable of detecting dozens of mRNA sequences for each initial droplet eluted from the sample. PUBLIC HEALTH RELEVANCE: We propose to develop an innovative integration of nanometer-resolution laser lysis and microfluidic tool to analyze single-cell heterogeneity in situ using qRT-PCR. In its ultimate implementation, users can simply load a tissue slice in the device, select the target cells based on their location and morphology, and then the system delivers mRNA expression of dozens of genes of individual cells in one hour.

描述（由申请人提供）：当前的单细胞 RT-PCR 分析严格限于分离细胞的分析，妨碍了其与细胞状态原位分析的兼容性，并且丢失了天然组织内初始细胞位置和形态的信息。我们建议开发一种基于微流体的创新工具，供临床和科学用户使用单细胞 mRNA 表达分析来原位分析基因表达异质性。该设备使用双光子激光连续裂解 3D 组织内已知坐标处的单个细胞。与传统的单光子激光裂解不同，双光子激光裂解依靠超快脉冲光源与生物材料之间的非线性相互作用，在纳米级聚焦体积内实现能量精确转移到细胞。裂解物立即转移至基于乳液（油滴）的 qRT-PCR 模块以分析 mRNA 表达。通过优化激光功率并使用具有精确流速控制的流体动力流动聚焦，可以最大程度地减少顺序裂解细胞之间的残留污染。由于微流通道的规模较小，样品处理的总体积通量减少至微升，细胞裂解和裂解液封装之间的时间间隔为秒量级，qRT-PCR 的完成时间约为一小时。该技术非常适合基础生物医学研究和临床应用，例如评估单细胞基因表达中的肿瘤细胞群异质性。此外，该技术还适合未来的发展，以增加可量化的基因数量。最终的实现将是一个高度多重化的平台，能够检测从样品中洗脱的每个初始液滴的数十个 mRNA 序列。 公共健康相关性：我们建议开发纳米分辨率激光裂解和微流体工具的创新集成，以原位分析单细胞异质性 使用 qRT-PCR。在其最终实现中，用户只需将组织切片加载到设备中，根据目标细胞的位置和形态选择目标细胞，然后系统在一小时内传递单个细胞的数十个基因的mRNA表达。