Multiplexed mRNA and miRNA Profiling of Single Cells Phase II

项目总结 该计划的第一阶段达到或超过了成功标准，实施了miRNA检测和 用2700代用信使核糖核酸分析方法直接对流式细胞仪进行单细胞分析 基因。已经分别证明了FFPE的TEMPO-SEQ分析与匹配的冰冻的相关性，这一阶段 II将讨论NHGRI特别感兴趣的主题F“单细胞基因组分析”，建立和 验证基于miRBase 21的整个miRNome Tempo-Seq分析，然后验证和 商品化单细胞Tempo-Seq Open App在测量人体的Fluidigm C1上的检测 完整转录组，补充了额外的临床相关的替代完整转录组 基因和突变，以及整个miRNome。这些分析将以散装细胞为基准 Tempo-Seq检测、C1/BioMark HD qPCR检测、C1Clontech Smarter NGS检测和RNAseq检测 大量的细胞。由于Fluidigm C1并不是唯一一台可以提供单电池的商用仪器 为了测试样品，我们还将开发和验证微流控仪器(细胞普查™)，以 使用FAC分离或在硅生物系统上制备的单个细胞进行TEMPO-SEQ分析 DEP阵列系统。使用DEP阵列，我们将演示分离的FFPE的单细胞分析。这个 对调查人员的意义在于，Tempo-Seq检测可以提供相同的覆盖范围和良好的或 测量RNAseq等低表达基因的灵敏度更高，但更多10到100个单细胞样本可以 以相同的成本进行测序，这一点很重要，因为可以运行更多的样本以提供更高的 高质量数据准确而灵敏的分子图谱可靠地识别单个细胞的亚型，尽管 在单细胞水平上随机基因表达的混杂影响，并促进单细胞研究 再快点。能够对FFPE执行单细胞分析，利用 DEPArray和Tempo-Seq将通过允许单细胞和 从FFPE存档样本中确定患者5、10、20年预后的亚群 都是已知的。

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.