Genome-wide, targeted expression analysis that combines the sensitivity of multiplex PCR with the dynamic range and throughput of NGS
The DriverMap™ Human Genome-Wide Targeted RNA Expression Profiling (EXP) Platform enables targeted multiplex PCR amplification and next-generation profiling to measure the expression levels of up to 19,000 human protein-coding genes or up to 5,000 mouse genes in a single assay. By combining highly multiplexed RT-PCR amplification with the depth and precision of next-generation sequencing (NGS) quantitation, the DriverMap EXP assay provides convenient, comprehensive, highly sensitive, and quantitative measurement of gene expression from as little as a few ng of total RNA.
Available off-the-shelf assay formats include the human genome-wide DriverMap EXP assay, DriverMap EXP for dried blood microsamples, or several panels of expertly curated gene targets. Cellecta also offers a DriverMap expression profiling service with optional downstream bioinformatics analysis.
Rather than reverse-transcribing the whole transcriptome, the DriverMap EXP assay combines multiplex RT-PCR to amplify defined and conserved 80- to 200- base regions of up to 19k human genes and then uses next-generation sequencing (NGS) to quantitatively assess abundance levels of each of these transcript amplicons.
There are significant advantages to targeting, amplifying, and sequencing carefully selected discrete loci of interest in the expressed transcriptome. Gene-specific primers that target each gene of interest obviate the need for RNA preparation to remove unwanted sequences such as ribosomal RNA, beta-globins, or other non-coding RNA. Only transcript sequences matching the targets of interest are amplified. Therefore, only a small amount of total RNA is needed for this highly sensitive assay. As little as a few ng of total RNA from single-cell lysate is enough to detect most target transcripts.
Also, the NGS read depth required to reliably profile several thousand targeted amplicons is much less than the depth required for the whole transcriptome. As a result, the targeted approach detects lower-abundance expressed transcripts more consistently and reliably than other RNA-seq approaches and much less sequencing depth is required.
Analysis of the sequencing results is much simpler with the DriverMap targeted approach since each amplicon corresponds to a known reference sequence. There is no need to estimate gene copy number from assembled cDNA fragments. With targeted RT-PCR the read levels of each amplicon directly correlate to the expression levels of the target transcript. The analysis can be done entirely on an Excel spreadsheet using housekeeping genes as references like standard qRT-PCR. Open-source Salmon software is recommended for use with the DriverMap EXP kit to enable this amplicon alignment and to generate the read counts for targets directly from the Illumina FASTQ file.
The DriverMap EXP assay starts with total RNA. The targeted, in-lab-validated primers used in the DriverMap EXP assay specifically amplify the target transcript amplicon sequences with minimal background from other non-target RNAs. As a result, total RNA can be assayed directly without the removal of rRNA or globin components. No mRNA enrichment is needed. Any standard RNA sample for RT-PCR is suitable for DriverMap EXP analysis, including total RNA from cells, frozen tissue, fine needle aspirate (FNA), whole blood, peripheral blood mononuclear cells (PBMCs), and mouse patient-derived xenograft (PDX) isolates. Cellecta also offers a DriverMap EXP profiling assay for use with dried blood microsamples collected using Neoteryx Mitra sampling tips for convenient and reliable sampling even from remote locations.
The top figure labeled "Donor #1" shows genes detected at time zero from two donors using the DriverMap EXP v3 assay collected using standard blood collection tubes (400 ul) vs Mitra tips (30 ul). Results show reproducible and sensitive expression profiling with >90% overlap in gene expression profiles, as well as strong correlation (r= 0.94) between expression levels obtained from Mitra microsamplers and phlebotomy-acquired samples.
In addition, the DriverMap EXP assay specifically amplifies human transcripts in a background of mouse or other non-human RNAs. This feature makes it an excellent choice for analysis of PDX samples or similar mixed samples with RNA from human and other organisms.
The bottom figure shows extremely sensitive detection of gene expression starting from very low levels of input total RNA in both HEK293 and Jurkat cells.
