Gene Expression Profiling of Single-Cell Samples: DriverMap Targeted Expression Profiling vs SMART Technology

Single-cell expression analysis provides insights about gene expression and cell heterogeneity at the single-cell level. It enables the elucidation of intracellular gene regulatory networks and intracellular pathways that would otherwise be masked in bulk analysis (Massaia et al., 2018). The DriverMap™ Targeted Gene Expression Profiling (EXP) assay combines highly multiplexed RT-PCR amplification with the depth and precision of Next-Generation Sequencing (NGS) to quantitatively measure gene expression of up to 19,000 target genes in a single assay–even down to the single-cell level.

The DriverMap EXP assay is an easy-to-run, single-tube, single-day assay that quantifies gene transcript levels directly from total RNA isolated from cells, tissue or blood, or from cellular extract using the DirectCell™ Protocol. No mRNA enrichment, or rRNA, mitochondrial, beta-globin depletion, or other processing is required. The single-tube assay provides robust, quantitative, and reproducible measurements of each expressed gene over as much as five-orders of magnitude differences in expression level.

The SMARTⓇ template-switch oligo approach to synthesize cDNA is often preferred for RNA sequencing of small samples and single cells since the approach is more specific for mRNA transcripts than standard poly-A or oligo-based reverse transcriptase. As a result, SMART-synthesized cDNA can be used directly for expression profiling without enriching for mRNA.

To compare head-to-head results between the DriverMap EXP approach–which also does not require mRNA enrichment–with the SMART template-switch oligo technology, the expression levels of 1,400 cancer-related genes (e.g. genes regulating DNA repair, angiogenesis, cell adhesion, etc.) targeted with the DriverMap™ Human Pan-Cancer Panel (96 multiplex) were assayed in single HCC1395 and HCC1395-BL cells.

The data in Figure 1 shows that DriverMap EXP detects ~ 20% more genes than SMART technology in single cells. Since the DriverMap technology is based on the hybridization of gene-specific primers with target RNA, the assay can be run directly from cell lysate without losing target mRNAs during RNA purification. Also, DriverMap uses robust reverse-transcription with random primers, instead of the 5’-transcript initiation utilized by the SMART-based first-strand synthesis. As a result, DriverMap EXP provides more comprehensive expression profiles from single cells than SMART-based approaches. Also, since the targeted primers are able to amplify gene transcripts directly from cell lysate without additional purification, it is a more convenient, and more robust profiling assay for FACS-sorted individual cells and small cell samples.

Figure 1: Genes detected using the SMART assay on the x-axis vs. the DriverMap assay on the y-axis where each dot represents the number of NGS reads (signal) for the target gene. Coordinate (0,0) represents genes that are not detected by either assay. The circle parallel to the y-axis (0 for x-coordinate) are genes that are not detected with the SMART assay whereas the few scattered genes across the bottom at the 0 y-coordinate are genes that are not detected with the DriverMap assay.

More information on the DriverMap Targeted Expression Profiling (EXP) Assay, which provides information on up to 19,000 human protein-coding genes or smaller specific, highly curated panels available here.

References:

Massaia, A., Chaves, P., Samari, S., Miragaia, R. J., Meyer, K., Teichmann, S. A., & Noseda, M. (2018). Single cell gene expression to understand the dynamic architecture of the heart. Frontiers in Cardiovascular Medicine, 5. https://doi.org/10.3389/fcvm.2018.0016

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