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.
This project will demonstrate the feasibility of a targeted sequencing assay, RASL-Seq, to measure mRNA and miRNA targets from single cells. 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, and will promote clinical research by enabling investigators to characterize rare cells, such as circulating tumor cells undergoing metastasis.
