Abstract
BackgroundWe have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.Methodology/Principal FindingsThe method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R2∼0.76–0.80 between individual cells and R2∼0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R2∼0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.Conclusions/SignificanceIn summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology.
Highlights
There has been growing interest in obtaining gene expression profiles from single cells, as it has become increasingly evident that the heterogeneity present in cell populations is such that population-based transcriptional profiles may not reflect the regulatory networks functional at the individual cell level [1,2]
We report on the comprehensive characterization of the performance of our single cell gene expression assay, termed Whole-Genome Gene Expression in Single Cells (WG-XSC), using picogram quantities of total RNAs, as well as a variety of different single cell types
We describe the utility of the WG-XSC assay in the transcriptional profiling of single cells and low input material, for which existing conventional methods are not sufficiently sensitive
Summary
There has been growing interest in obtaining gene expression profiles from single cells, as it has become increasingly evident that the heterogeneity present in cell populations is such that population-based transcriptional profiles may not reflect the regulatory networks functional at the individual cell level [1,2]. Apart from facilitating cell lineage mapping an additional key utility of single cell transcriptomics is in clinical diagnostics, the identification of gene expression signatures in circulating tumor cells for use as prognostic markers for metastatic tumors [17] and treatment response [18]. While there have been several recent advancements in the capture and isolation of single cells, such as cell picking [19,20] and microfluidic [1,17,21] devices, there remains a need for the development of high-throughput, whole-genome gene expression assays for single cells. We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs
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