5 min readFaster, simpler and more sensitive technology for biomarker discovery in total RNA samples

Transcriptome snapshots can provide invaluable insights into gene expression patterns, help infer cell activity in a sample, identify novel drug targets and measure the expression of important disease biomarkers.

Latest transcriptome-based profiling approaches, such as RNA-sequencing (RNAseq), have revolutionized diagnostics and biomarker discovery, allowing researchers to easily obtain expression snapshots of whole genomes.

However, the majority of current techniques have high sample requirement, poor sensitivity and specificity as well as limited throughput. The fast-growing field of biomarker discovery requires new sophisticated tools for the investigation of complex biological samples.

The DriverMap™ Human Genome-Wide Targeted Expression Profiling Assay, developed by Cellecta, is a novel, comprehensive, whole-genome targeted expression profiling solution intended for research applications. 

The DriverMap™ assay is based on a targeted RNA expression profiling platform that produces more sensitive and comprehensive results than current methods relying on array hybridization or RNA-seq.

The assay enables researchers to simultaneously measure the expression level of almost 19,000 human protein-coding genes in a single tube. 

By combining highly multiplexed RT-PCR amplification with the depth and precision of next-generation sequencing (NGS) quantitation, the DriverMap™ assay provides convenient, comprehensive, highly sensitive, and quantitative measurement of gene expression from total RNA.

Moreover, unlike most expression analysis approaches, the DriverMap™ assay starts with total RNA and does not require ribosome depletion or mRNA enrichment.

The key features of the DriverMap™ assay are: 

  • Validated Primer Design. Each of the multiplex PCR primers are functionally validated in the laboratory. Cellecta’s unique multiplex primer design minimizes primer dimerization and cross-reactivity while maximizing specificity and efficacy.
  • High-Throughput Capabilities. The assay enables expression profiling of thousands of genes in a single-tube reaction.
  • Exquisite Sensitivity, Specificity & Reproducibility.  DriverMap™ allows low abundance transcripts to be identified to facilitate biomarker discovery.
  • Broad Linear Dynamic Range. This makes characterization of all major immune cell types, and detection of infiltrating immune cells possible.
  • Modular and Flexible. Fully customizable panels to target a selection or all protein-coding genes are available, with simpler data analysis requirements than RNA-Seq.

Cellecta offers researchers access to the DriverMap™ assay as a kit to enable you to run the assay in your lab, and as a service if you prefer to send your samples to Cellecta for targeted expression profiling. 

The DriverMap™ workflow

The DriverMap™ technology 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.

The assay is based on a targeted RNA expression profiling platform that produces more sensitive and comprehensive results than current methods relying on array hybridization or RNA-sequencing (RNA-seq). It has broad sample compatibility and requires low input of total RNA.

The scheme below (Figure 1) demonstrates the DriverMap™ assay workflow.

The protocol involves a rapid and convenient, single-tube, RT-PCR directly followed by NGS (without intermediate steps) that provides robust measurements of each expressed gene from total RNA.

Figure 1: DriverMap Assay workflow combines the power of sophisticated primer design, multiplex RT-PCR and next-generation sequencing to yield a simple yet highly sensitive, reproducible targeted expression profiling solution.

The assay can be successfully performed with RNA levels as low as 10 pg (single-cell levels) and yields a highly quantitative and reproducible NGS readout, generating up to 5-orders of magnitude digital expression level measurement (Figure 2).

Figure 2: DriverMap™ Assay is able to measure expression from as low as 10 pg total RNA. The correlation (R-squared values) of detected genes between human universal RNA and total brain RNA remains highly consistent across a 10 pg to 50 ng of starting total RNA.

Learn more about DriverMap™ Assay Service on the Cellecta website.

Primer design and functional validation 

Key to the DriverMap™ approach is the efficiency and specificity of the multiplex PCR target amplification step.

Numerous factors–including secondary structure of target transcripts, 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.

Development of the DriverMap™ assay required sophisticated primer design work and an iterative, empirical testing process. For this, the company developed a high-throughput bioinformatics pipeline and carried out experimental validation of thousands of PCR primers in a highly complex multiplex reaction (Figure 3) from which they generated an optimized genome-wide, multiplex PCR assay for all 19,000 human protein-coding genes.

Figure 3: Development of Functionally Validated RT-PCR Primers for the DriverMap™ Assay

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). Primers are balanced for melting temperature and cross-dimers eliminated. 

A simple informatics screen was not sufficient to ensure effective and specific primers suitable for a comprehensive multiple PCR reaction. To address this, the Cellecta team had to test the performance of each primer set experimentally. 

For each target mRNA, they 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 (Figure 4), 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, the researchers selected primers with low specific efficacy, which allowed them to solve the problem of over-sequencing highly expressed target transcripts.

Figure 4: Experimental Validation Yields a Selection of Optimized, Highly Specific Primers for the DriverMap™ Assay

Learn more about DriverMap™ Assay Kit on the Cellecta website

Benefits of DriverMap™ over other methods

The following infographic shows how the DriverMap™ Assay compares to the ‘gold standard’ of gene expression profiling, RNAseq technology. 

infographic updated

DriverMap™ Assay is faster, simpler and more sensitive than the targeted RNAseq.

The Assay has a wider dynamic range and 10- to 100-fold greater sensitivity than microarray and RNA-seq expression analysis methods, which allows to detect more low- and medium-abundant transcripts (Figure 5).

Figure 5: RNA-Seq vs DriverMapTM Assay Heatmap. DriverMapTM Targeted Expression Profiling assay is 100-fold more sensitive than RNA-Seq. With a broader dynamic range than RNA-Seq, at the high-abundant expression levels, differentially expressed abundant genes are less compressed so differences can be better detected. On the low-abundant levels, increased sensitivity detects more low-abundant genes.

DriverMap™ Assay Kit can be used with total RNA from any source

Unlike most expression analysis approaches, the DriverMap™ assay starts with total RNA. No mRNA enrichment is needed. 

Any standard RNA sample for RT-PCR is suitable for DriverMap™ analysis, e.g. total RNA from cells, frozen tissue, fine needle aspirate (FNA), whole blood, peripheral blood mononuclear cells (PBMCs), and mouse patient-derived xenograft (PDX) isolates.

The targeted, validated primers used in the DriverMap™ assay specifically amplify the target transcript amplicon sequences with minimal background from other non-target RNAs. As a result, total RNA from blood can be assayed directly without removal of rRNA or globin components.

In addition, with the DriverMap™ assay, human transcripts can be specifically amplified against the background of mouse RNAs in PDX samples. 

DriverMap™ Assay has a wide range of applications

Some cases where DriverMap™ Assay would be the technology of choice due to its simple, more efficient workflow and greater sensitivity:

  • Biomarker Discovery from Whole Blood Samples — whole blood RNA (e.g., PAXgene) can be used without previous globin and ribosomal RNA depletion.
  • Circulating Tumour Cell (CTC) Detection in Whole Blood — no enrichment step needed.
  • Tumour Cellular Composition Analysis — the assay yields comprehensive expression profiles to assess tumour/stromal cell content and profile infiltrating immune cell types.
  • Patient-Derived Xenograft (PDX) Models — human-specific expression profiles can be obtained without separation of human cells from model-animal background cells.
  • Expression Analysis of Cell Lines — easy 96- or 384-well single-tube DriverMap™ protocol facilitates identification of the mechanisms of chemical and genetic perturbations.
  • Expression Profiling of Rare and Limited Cell Populations — DriverMap™ allows comprehensive analysis with limited numbers of cells isolated by FACS, magnetic beads or microfluidics, fine needle biopsies, and laser capture microdissection.

DriverMap™ assay can be ordered as a kit to enable you to run the assay in your lab, and as a service if you prefer to send your samples to Cellecta for targeted expression profiling.

Analytical method, Biomarker discovery, Cellecta, Cellular composition analysis, CTC detection, Gene expression, Genomic Sequencing, Microarray, molecular biology, mRNA, RT-PCR, Targeted expression profiling, Transcriptomics, Whole blood profiling

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