Multiplexed mRNA and miRNA Profiling of Single Cells

描述（由申请人提供）：该I期项目将展示RASL-Seq靶向测序测定的可行性，用于测量单细胞中的mRNA和miRNA靶点。RASL-Seq是一种基于连接的测序读出检测，类似于Illumina上市的检测，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，这是单细胞分析的一个关键优势。RASL-Seq已经在基于微滴板细胞的表达筛选中成功地分析了mrna，用成对的25-mer探针靶向裂解物中的mrna，这些探针经过杂交、连接和扩增，连接到测序适配器和条形码上，以识别每个单独的样品。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分析的障碍是短（20至22 nt） miRNA序列的测量，高度同源miRNA的分化，在小体积中进行分析以最大化灵敏度，以及实现单细胞灵敏度。重大变化

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.