TempO-LINC high throughput, high sensitivity single cell gene expression profiling assay

TempO-LINC 高通量、高灵敏度单细胞基因表达谱分析

• 批准号: 10156786
• 负责人: BRUCE E. SELIGMANN
• 金额: $ 40.23万
• 依托单位: BIOSPYDER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10156786
• 关键词: Address; Archives; Bar Codes; Benchmarking; Biological Assay; Biological Markers; Cell Line; Cells; Cellular Assay; Complementary DNA; Coupling; Crosslinker; Data; Disease; Dose; Gene Expression; Gene Expression Profiling; Gene Fusion; Genes; Genomics; Goals; Human; In Situ; In Situ Hybridization; Ligase; Ligation; Measurement; Measures; Methods; Molecular; Mouse Cell Line; Mus; Noise; Normal Statistical Distribution; Nucleotides; Oligonucleotides; Pathway interactions; Performance; Peripheral Blood Mononuclear Cell; Phase; Polyadenylation; Process; Protocols documentation; RNA; RNA Splicing; Research Personnel; SELL gene; Sampling; Signal Transduction; Single Nucleotide Polymorphism; Sorting - Cell Movement; Species Specificity; Specificity; Sulfhydryl Compounds; Sum; Suspensions; Time; Tissues; Transcript; Untranslated RNA; Variant; adduct; base; cell preparation; combinatorial; cost; crosslink; data quality; detector; differential expression; drug development; drug efficacy; experimental study; improved; innovation; medication safety; novel; prevent; programs; response; safety assessment; single cell sequencing; tool; transcriptome; transcriptome sequencing; translational medicine

Summary/Abstract: Single cell gene expression assays have become important tools to identify functional subtypes of cells, as well as changes within specific subtypes resulting from diseases or treatments. However, most current methods require dedicated hardware and kits, making them expensive, and most are 3’ based and thus cannot measure splice variants, gene fusions, expressed single nucleotide variants, or RNAs that are not polyadenylated. Furthermore, there is no method to-date that enables investigators to measure low or even many moderately expressed genes from single cells, which prevents measurements of many key biomarkers and important genes in molecular pathways and functions. Current methods also only measure a limited number of genes/cell and do not provide quantitative measurements of the abundance of those genes, and thus cannot be used to measure changes in expression level along the order of magnitude possible using bulk preparations of cells, nor can they be used to carry out dose response experiments at the single cell level. Our approach will enable investigators to carry out single cell assays without purchase of proprietary hardware, and provide a level of performance comparable to the profiling of bulk cell samples. Based on the commercial targeted gene expression TempO-Seq® bulk cell assay, we will implement a TempO-LINC single cell assay which, based on preliminary data, will provide data measuring the same genes (low, moderate, high expressed) and similar number of genes/cell as can be measured from bulk samples, quantitatively, providing a similar dynamic expression range and normal distribution of counts as measured from bulk samples. TempO-LINC can be used to process a few 100 single cells up to 100,000+ cells at a time. TempO-LINC data will be benchmarked against both bulk cell data and 10x Genomics single cell data. Furthermore, we will demonstrate utility to measure splice variants and to provide single cell dose response data. These data will be generated using the commercial S1500 surrogate whole transcriptome assay adapted to the TempO-LINC process. In a follow-on Phase II program, the human and mouse whole transcriptome assays will be implemented, the TempO-LINC assay commercialized, and additional applications demonstrated using cell lines, purified cells, and cells dissociated from tissues. The performance and capabilities of the TempO-LINC single cell assay will drive expansion of applications pursued within the single cell field beyond identification of subpopulations of cells.

摘要/摘要：单细胞基因表达测定已成为识别功能的重要工具 细胞的亚型以及由疾病或治疗引起的特定亚型的变化。然而， 大多数当前方法都需要专用的硬件和套件，使其昂贵，大多数是基于3'的，并且 因此无法测量剪接变体，基因融合，表达的单个核苷酸变体或不是RNA 聚腺苷酸化。此外，没有任何方法可以使研究人员能够测量低甚至许多 来自单细胞的中等表达基因，可防止许多关键生物标志物的测量 分子途径和功能中的重要基因。当前方法还仅测量有限数量的 基因/细胞，不提供这些基因抽象的定量测量，因此不能是 用于测量沿着大小准备的数量级的表达水平的变化 细胞，它们也不能用于在单细胞水平上进行剂量反应实验。我们的做法意愿 使调查人员能够在不购买专有硬件的情况下进行单细胞分析，并提供一个水平 性能与散装细胞样品的分析相当。基于商业靶向基因 表达tempo-seq®Bulk细胞测定法，我们将实施一个tempo-linc单细胞测定法，该测定法基于 初步数据将提供测量相同基因（低，中等，高表达）和相似的数据 可以从批量样品中测量的基因/细胞数量，并提供类似的动态 表达范围和计数的正态分布，如大量样品测量。可以使用tempo-linc 一次处理几个最多100,000多个细胞的单个单元。 TEMPO-LINC数据将基准针对 散装细胞数据和10倍基因组学单细胞数据。此外，我们将展示测量剪接的实用性 变体并提供单细胞剂量响应数据。这些数据将使用商业S1500生成 替代整个转录组测定法适应了速度-linc工艺。在后续第二阶段计划中， 人和鼠标将实施整个转录组分析，速度-linc分析商业化， 以及使用细胞系，纯化的细胞和与组织分离的细胞证明的其他应用。 TEMPO-LINC单细胞测定的性能和功能将推动追求的应用程序的扩展 在单细胞场中，超出了细胞亚群的鉴定。