As our understanding of the human genome starts to yield more information about the interplay of genes in disease progression and development, the ability to multiplex quantitative nucleic acid analysis technologies has become a goal for technological development, reported Jim Thorn, Ph.D., European bioseparations product manager at Beckman Coulter. “While qPCR has remained the backbone of microarray validation studies and SNP detection research, its drawback is that it can only handle one gene per test, and multiplexing the qPCR process has so far remained elusive, ” said Dr. Thorn.
For studies where only a few genes or transcripts need to be analyzed, the one-gene-one-assay approach may not be such a problem, but when large numbers of genes need to be evaluated in copious samples, the costs can escalate. “High-throughput qPCR instrumentation is now standard in many large laboratories, but if the requirement is to look at a signature of, for example, 30 genes in 1,000 samples, you’d need to run 30,000 tests, and then triple that to carry out three technical replications. The costs in terms of reagent requirements can become untennable.”
To address these issues, Beckman Coulter has commercialized a capillary electrophoresis-based system, the GenomeLab GeXP genetic analysis system, as a quantitative platform that utilizes a patented, multiplexed reverse transcription PCR (RT-PCR) approach to investigate multiple genes or gene sets.
The GenomeLab GeXP is essentially an endpoint PCR reaction coupled with a separation technology, which can run up to 40 genes per sample for gene expression, Dr. Thorn explained. “It’s a capillary electrophoresis sequencer, enabled to separate gene-expression samples. So when you run the assay you see a separate signal for each gene. For applications in gene-expression analysis, we supply the user with the GeXP software to design multiplex assays and the relevant GeXP kits for gene-specific reverse transcription and PCR amplification of their target mRNA from 25 ng of total RNA.”
The multiplexed endpoint PCR reaction is carried out on a standard PCR platform. This part of the technology hinges on a universal priming strategy, such that the PCR reaction uses four primer types. Two per gene are the forward and reverse gene-specific primers carrying a universal tail, which the customer obtains from a preferred provider. The other two primers are universal primers that bind to those tails and are the same for all genes in the multiplex.
“The chemistry is set up so there is a huge excess of the universal primers. This means that for the first one or two PCR cycles the gene-specific primers kick in, and after that the universals take over. Effectively this results in a PCR reaction that locks the ratios of the genes at an early stage, so when the universal primers take over replication, the target sequences are always copied in the same ratio. We have a reaction that behaves as if it’s one single type of PCR product, even though there could be up to 40 or so different genes being amplified.”
After amplification, a small sample of the product is transferred into a 96-well plate for separation and analysis in the GenomeLab GeXP. “Importantly, while microarray and qPCR-based technologies can typically investigate twofold or greater changes in gene expression, the GenomeLab GeXP technology can reliably detect expression changes down to as low as 0.5-fold,” Dr. Thorn claimed.