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: 8551657
• 负责人: Deirdre R. Meldrum
• 金额: $ 19.62万
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8551657
• 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; public health relevance; response; sample collection; single cell analysis; time interval; tool; tumor; two-photon; 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; public health relevance; 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.

描述（由申请人提供）：当前对单细胞的RT-PCR分析严格限于分解相关细胞的分析，以防止其与细胞状态的原位analsis的兼容性，并失去天然组织中初始细胞位置和形态的信息。我们建议使用单细胞mRNA表达分析来开发一种创新的基于微流体的工具，以原位分析基因表达异质性。该设备使用两个光子激光器在3D组织内的已知坐标处串行裂解单个细胞。与常规的单光子激光裂解不同，两光子激光裂解依赖于超快脉冲光源与生物材料之间的非线性相互作用，以实现精确地转移到纳米尺度焦点内的细胞中。立即将裂解物转运到基于乳液的（油-DROPLET）QRT-PCR模块中，以配置mRNA表达。通过优化激光功率和使用精确流速控制的流体动力流动聚焦来最大程度地降低依次裂解细胞之间的污染。由于微流体通道的尺度很小，样品处理的总体积通量减小到微量流量，因此细胞裂解和裂解物封装之间的经过的时间间隔在秒的顺序上，QRT-PCR的完成量为一小时。该技术非常适合基本的生物医学研究和临床应用，例如评估单细胞基因表达中肿瘤细胞群异质性。此外，该技术还适合未来的发展，以增加可以量化的基因数量。最终的实现将是一个高度多重的平台，能够检测从样品中洗脱的每个初始液滴的数十个mRNA序列。