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Multiplexed mRNA and miRNA Profiling of Single Cells Phase II

单细胞 II 期多重 mRNA 和 miRNA 分析

基本信息

批准号：
9356539
负责人：
BRUCE E. SELIGMANN
金额：
$ 49.89万
依托单位：
BIOSPYDER TECHNOLOGIES, INC.
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：
2014-07-18 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9356539
关键词：
Address Aging Archives Area Benchmarking Biological Assay Biological Models Breast Cancer cell line Budgets Cell Separation Cells Clinical Computer Simulation Contracts Data Data Quality Development Family Freezing Gene Expression Gene Mutation Genes Genomics Human Hybrids Immune Immunology Immunophenotyping Ligation MCF7 cell Malignant Neoplasms Marketing Measures Mental Health Messenger RNA Methods MicroRNAs Microfluidics Modeling Modification Molecular Profiling Mutation National Human Genome Research Institute Neurosciences Oligonucleotides Outcome Patients Performance Phase Phenotype Process Production Protocols documentation RNA Reproducibility Research Research Personnel Running Salts Sampling Silicon Sorting - Cell Movement Specificity System Testing Time Tissues Transcript attenuation base cell preparation clinically relevant cost design detector experimental study fusion gene innovation instrument interest malignant breast neoplasm molecular phenotype performance tests programs prototype single cell analysis success targeted sequencing transcriptome transcriptome sequencing translational medicine tumor

项目摘要

PROJECT SUMMARY The Phase I of this program met or exceeded the success criteria, implementing a miRNA assay and directly demonstrating single cell analysis on the Fluidigm C1 using a surrogate mRNA assay of 2700 genes. Having separately shown that TempO-Seq assay of FFPE correlates to matched frozen, this Phase II will address an NHGRI area of special interest Topic F “Single cell genomic analysis”, establishing and validating a whole miRNome TempO-Seq assay based on miRBase 21, then validating and commercializing single cell TempO-Seq Open App assays on the Fluidigm C1 that measure the human whole transcriptome, a surrogate whole transcriptome supplemented with additional clinically relevant genes and mutations, and the whole miRNome. These assays will be benchmarked against bulk cell TempO-Seq assay, C1/BioMark HD qPCR assay, C1 Clontech SMARTer NGS assays, and RNAseq assay of bulk cells. Since the Fluidigm C1 is not the only commercial instrument that can provide single cell samples for testing, we will also develop and validate a microfluidic instrument (the CellCensus™) to perform the TempO-Seq assay using single cells separated by FACS or prepared on Silicon Biosystems DEPArray system. Using the DEPArray we will demonstrate the single cell assay of dissociated FFPE. The significance to investigators is that the TempO-Seq assays can provide the same coverage and as good or better sensitivity to measure low expressed genes as RNAseq but 10 to 100 more single cell samples can be sequenced for the same cost, which is important because more samples can be run to provide higher quality data accurate and sensitive molecular profiles to reliably identify subtypes of single cells despite the confounding impact of stochastic gene expression at the single cell level, and advance single cell research faster. The ability to perform single cell analysis on FFPE, exploiting the unique advantages of the DEPArray and TempO-Seq, will advance translational medicine by permitting the assay of single cells and identification of subpopulations from archived FFPE samples for which patient 5-, 10-, 20-year outcomes are known.

项目摘要该项目的第一阶段达到或超过了成功标准，实施了miRNA测定，使用2700的替代mRNA测定直接证明了Fluidigm C1上的单细胞分析基因.已经单独显示FFPE的TempO-Seq测定与匹配的冷冻相关，该阶段 II将解决一个NHGRI特别感兴趣的领域主题F“单细胞基因组分析”，建立和基于miRBase 21验证整个miRNome TempO-Seq测定，然后验证和在Fluidigm C1上商业化单细胞TempO-Seq Open App测定，全转录组，一个替代的全转录组，补充了其他临床相关的基因和突变，以及整个miRNome。这些测定将以散装细胞为基准 TempO-Seq检测试剂盒、C1/BioMark HD qPCR检测试剂盒、C1 Clontech SMARTer NGS检测试剂盒和RNAseq检测试剂盒散装细胞。由于Fluidigm C1不是唯一可以提供单细胞的商业仪器，我们还将开发和验证微流体仪器（CellCensus™），使用通过FACS分离或在Silicon Biosystems上制备的单细胞进行TempO-Seq测定 DEPAray系统。使用DEPArray，我们将证明解离的FFPE的单细胞测定。的对研究者的重要性在于，TempO-Seq测定可以提供相同的覆盖率，并且与传统的检测方法一样好，更好的灵敏度来测量低表达的基因作为RNAseq，但10至100多个单细胞样品可以这一点很重要，因为可以运行更多的样本，以提供更高的高质量的数据准确和敏感的分子谱，以可靠地识别单细胞的亚型，单细胞水平随机基因表达的混杂影响，并推进单细胞研究快在FFPE上进行单细胞分析的能力，利用 DEPArray和TempO-Seq将通过允许单细胞测定来推进转化医学，从存档FFPE样本中识别亚群，患者5年、10年、20年结局是已知的。