Multiplexed mRNA and miRNA Profiling of Single Cells Phase II

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 C using a surrogate mRNA assay of 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 then validating and commercializing single cell TempO Seq Open App assays on the Fluidigm C 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 C BioMark HD qPCR assay C Clontech SMARTer NGS assays and RNAseq assay of bulk cells Since the Fluidigm C 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 to 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 year outcomes are known PROJECT NARRATIVE Having met or exceeded the Phase I success criteria implementing a miRNA assay and directly demonstrating single cell analysis on the Fluidigm C using a surrogate mRNA assay of genes this Phase II will address an NHGRI area of special interest Topic F Single cell genomic analysis by implementing an assay of the whole miRNome validating and marketing C Open App assays of the whole miRNome whole transcriptome and surrogate whole transcriptome and fabricating and validating a microfluidic CellCensus platform to perform these same TempO Seq assays on single cells from FACS sorting and DEPArray including from dissociated FFPE using the DEPArray The TempO Seq assay will permit investigators to assay to times more samples then they can today using RNAseq with high sensitivity to measure low expressed genes for the same sequencing cost accelerating the rate of progress in single cell research and the quality of data Developing the CellCensus will make TempO Seq single cell analysis available to all investigators not just those with a C and will make possible efficient assay of single cells from FFPE using the DEPArray advancing translational medicine by permitting the assay of single cells and identification of subpopulations from archived FFPE samples for which patient year outcomes are known