Targeted Expression Profiling: Driver-Map™ Assay
The Driver-Map™ assay is based on an targeted RNA expression profiling platform that produces more sensitive and comprehensive results than current methods relying on array hybridization or RNA-sequencing (RNA-seq). The Driver-Map method combines multiplex RT-PCR amplification to amplify a defined and conserved 80-250 base segment in the transcript of each targeted gene, with quantitative analysis by next-generation sequencing (NGS) to assess abundance levels for each of the amplified transcript amplicons.
Key to the Driver-Map approach is the efficiency and specificity of the multiplex PCR target amplification step. Numerous factors–including secondary structure, non-specific binding, primer binding inefficiency and primer-primer interactions, which may be tolerated in a simple PCR reactions–can cause significant problems in a multiplex PCR environment, particularly when one attempts to amplify thousands of templates in the same reaction.
Sophisticated Primer Design and Functional Validation Yield Optimized Primer Sets for Multiplex PCR Reaction
Development of the Driver-Map assay required sophisticated primer design work and an iterative, empirical testing process. For this, we developed a high-throughput bioinformatics pipeline and carried out experimental validation of thousands of PCR primers in a highly complex multiplex reaction (Fig. 1) from which we generated an optimized genome-wide, multiplex PCR assay for all 19,000 human protein-coding genes.
Primers were initially selected and screened for several characteristics. For example, all primers are selected to have GCA-rich sequences (with minimum T content) in order to reduce primer dimer formation in multiplex PCR assay. In addition, primers are selected for high specificity (more than two mismatches for any other gene including rRNA, repetitive elements, and globin RNAs), high Tm (more than 63 °C), and a small size of amplicon (80-250-n).
A simple informatics screen was not sufficient to ensure effective and specific primers suitable for a comprehensive multiple PCR reaction. To address this, we had to test the performance of each primer set experimentally. For each target mRNA, we synthesized the best 5–20 PCR primers then ran them in a several multiplex RT-PCR reactions using a set of universal control human tissue/cell line RNAs (Fig. 2), mouse negative control RNAs and positive control DNA as templates. Only one set of primers targeting conservative portions of different mRNA isoforms (usually 3′-ORF region) with the highest efficiency, sensitivity, and specificity was selected for each mRNA. Furthermore, for highly abundant transcripts, we selected primers with low specific efficacy, which allowed us to solve the problem of over-sequencing highly expressed target transcripts.
Ultra-Sensitive, Specific Assay Effectively Targeting 19,000 Human Protein-Coding Genes in a Single Experiment
As a result of this meticulous process, we generated a complete set of validated multiplex RT-PCR primers providing reproducible amplification of all 19,000 human protein-coding transcripts (one cDNA fragment per gene) directly from total RNA. mRNA enrichment is not needed and, for blood samples, no globin depletion is required. NGS of the amplified products is then carried out to quantify the relative levels of each transcript using the targeted amplicons as a reference for sequencing deconvolution.
The result is a highly quantitative, targeted expression profiling assay that:
- measures levels of gene expression with great reproducibility (R-squared values > 0.92)
- requires only total RNA as input for a multiplex PCR reaction so it is simple and fast (1-day) to run
- generates defined single-amplicon sequencing results per gene, which makes deconvolution straightforward and easily comparable across samples
Cellecta offers researchers access to the Driver-Map assay as a kit to enable you to run the assay in your lab, and as a service if you prefer to send us your samples for targeted expression profiling.