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Kenneth James Livak Fluidigm Corporation, United States of America |
Abstract
The stochastic nature of generating eukaryotic transcripts challenges conventional methods for obtaining and analyzing single-cell gene expression data. As part of a study exploring the effect of genotype on Wnt pathway activation, data were collected for 96 qPCR assays on individual cells from 15 genotyped lymphoblastoid cell lines (HapMap lines). For each cell line, qPCR was used to obtain single-cell gene expression profiles for 48 baseline cells and 48 stimulated cells. Thus, data were collected from a total of 1440 single cells. The methods used in the collection and analysis of single-cell qPCR data will be contrasted with those used in conventional qPCR. The generation of RNA libraries from hundreds to thousands of single cells has been simplified by introduction of the C1™ Single-Cell Autoprep System (Fluidigm). This system captures up to 96 single cells and performs the processing steps of cell lysis, cDNA synthesis by reverse transcriptase, and initial amplification to generate libraries for qPCR or RNA-Seq analysis.
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