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Svilen Tzonev Digital Biology Center, Bio-Rad, United States of America |
Abstract
Single-cell transcript profiling is undoubtedly the ideal approach for gene expression analysis in heterogenous cell types. However, the robust detection of transcripts in isolated single cells is technically challenging, especially without pre-amplification. Droplet Digital PCR (ddPCR) developed at Bio-Rad’s Digital Biology Center directly counts individual molecules with superior precision and reproducibility. The ddPCR-based single-cell gene expression protocol measures even very low abundance transcripts with minimal sample processing for defined targets. Furthermore, ddPCR is performed in 96-well plates and is well suited to high throughput studies of focused sets of genes in large numbers of single cells.
In this work, we demonstrate the single-cell gene expression analysis of pluripotent P19 cells before and after neural induction. We present a simple and robust workflow for profiling multiplexed, transcript targets in flow-sorted, single cells. We characterize a panel of validated assays targeting stem cell, proliferation and differentiation marker genes including Sox2, Ki67, and EphrinB1, respectively. We compare expression levels of these genes in Retinoic Acid treated and not treated single cells and bulk RNA preparation from the same cell populations prior to sorting. We demonstrate that ddPCR provides absolute counts of transcripts from several thousand copies to less than ten copies per cell. Our findings are discussed with current data in the literature.
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