Multiplexed mRNA and miRNA Profiling of Single Cells

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, similarto 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 volum