RNA-Seq following PCR-based sorting reveals rare cell transcriptional signatures

Abstract

BackgroundRare cell subtypes can profoundly impact the course of human health and disease, yet their presence within a sample is often missed with bulk molecular analysis. Single-cell analysis tools such as FACS, FISH-FC and single-cell barcode-based sequencing can investigate cellular heterogeneity; however, they have significant limitations that impede their ability to identify and transcriptionally characterize many rare cell subpopulations.ResultsPCR-activated cell sorting (PACS) is a novel cytometry method that uses single-cell TaqMan PCR reactions performed in microfluidic droplets to identify and isolate cell subtypes with high-throughput. Here, we extend this method and demonstrate that PACS enables high-dimensional molecular profiling on TaqMan-targeted cells. Using a random priming RNA-Seq strategy, we obtained high-fidelity transcriptome measurements following PACS sorting of prostate cancer cells from a heterogeneous population. The sequencing data revealed prostate cancer gene expression profiles that were obscured in the unsorted populations. Single-cell expression analysis with PACS was subsequently used to confirm a number of the differentially expressed genes identified with RNA sequencing.ConclusionsPACS requires minimal sample processing, uses readily available TaqMan assays and can isolate cell subtypes with high sensitivity. We have now validated a method for performing next-generation sequencing on mRNA obtained from PACS isolated cells. This capability makes PACS well suited for transcriptional profiling of rare cells from complex populations to obtain maximal biological insight into cell states and behaviors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2694-2) contains supplementary material, which is available to authorized users.

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“…32 New droplet microfluidic techniques allow this using single-cell PCR, filling a major need, but are challenging to implement due to the requisite complex, multi-step and multi-device workflows. 33 Our results demonstrate an integrated on-chip solution for cell lysis and reagent merger, providing a faster and more reliable method for single-cell droplet RT-PCR.…”

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“…The FRISCR method[50] has great potential, but relies on suitable antibodies and known cell type markers. Promising alternatives include using fluorescent in situ hybridization or RT-PCR reactions to sort cells on the abundance of mRNA transcripts[58,59], and merging these methods with droplet-based scRNA-seq will be hugely advantageous.In the model of Patch-seq, protocols are sorely needed for sequencing RNA from single cells previously or concurrently characterized by other methods, e.g., physiology, connectivity, developmental lineage, or live imaging. Methods to either maintain[60] or reconstruct[61,62] spatial information in conjunction with scRNA-seq need further development for application to mammalian brain studies.…”

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