Multiplexed mRNA and miRNA Profiling of Single Cells

描述（由申请人提供）：本I期项目将证明靶向测序测定法RASL-Seq测量单细胞mRNA和miRNA靶标的可行性。RASL-Seq是一种基于连接的测定，具有测序读出，类似于Illumina销售的测定，但能够使用样品裂解物测量基因表达，而无需提取或逆转录RNA，这是单细胞分析的关键优势。RASL-Seq已经成功地在微量滴定板基于细胞的表达筛选中分析了mRNA，用成对的25-mer探针靶向裂解物中的mRNA，所述探针被杂交、连接和扩增，以连接识别每个单独样品的测序接头和条形码。目标复杂度范围从100秒到1000秒，允许通过查找表而不是转录组比对进行快速和简单的数据分析。miRNA分析的障碍是测量短（20至22 nt）miRNA序列，区分高度同源的miRNA，在小体积中进行测定以最大化灵敏度，以及实现单细胞灵敏度。显著变化 需要进行方案和探针设计，以测量单细胞分析所需的小体积的miRNA，但是与其它市售的测定不同，miRNA和mRNA可以同时测量，允许将miRNA水平标准化为mRNA管家，以及测量mRNA和miRNA的复杂特征。通过靶向测序和样本合并，RASL-Seq将从根本上降低相对于全转录组方法的测序成本/样本，同时提供单细胞中mRNA和miRNA的高度多重分析。

DESCRIPTION (provided by applicant): This Phase I program will demonstrate the feasibility of a targeted sequencing assay, RASL-Seq, to measure mRNA and miRNA targets from single cells. RASL-Seq is a ligation based assay with a sequencing read-out, similar to assays marketed by Illumina, but able to measure gene expression using sample lysates without the need to extract or reverse transcribe RNA, a key advantage for single cell analyses. RASL-Seq has successfully profiled mRNAs in microtiter plate cell-based expression screens, targeting mRNAs in lysates with pairs of 25-mer probes, which are hybridized, ligated, and amplified, to attach sequencing adapters and barcodes that identify each individual sample. Target plexity ranges from 100s to 1000s, permitting rapid and simple data analysis by lookup table rather than transcriptome alignment. Hurdles to miRNA analysis are measurement of short (20 to 22 nt) miRNA sequences, differentiation of highly homologous miRNAs, performing the assay in a small volume to maximize sensitivity, and achieving single cell sensitivity. Significant changes in the protocol and probe design need to be made to measure miRNA in the small volumes required for single cell analysis, but miRNA and mRNA can be measured at the same time, unlike other commercially available assays, permitting normalization of miRNA levels to mRNA housekeepers, as well as measurement of complex signatures of mRNA and miRNA. By targeted sequencing and sample pooling, RASL-Seq will radically decrease the cost/sample of sequencing relative to whole transcriptome approaches, while offering highly multiplexed analysis of both mRNA and miRNA in single cells.