Hybridization microarrays, which have emerged as the leading quantitative tool for analyzing transcription of many thousands of genes in a sample simultaneously, have a number of limitations in analytical performance and sample throughput.
Real-time or qPCR technology has emerged as the superior alternative because of its high accuracy, precision, and dynamic range. As a consequence, it is the reference assay for calibration and validation of microarray data. Scaling qPCR to analyze larger numbers of genes and samples simultaneously, however, is intrinsically prohibited by the logistics and cost of the assay in its current microliter format in 96- or 384-well microplates.
The recent “qPCR Symposium USA,” convened in Palo Alto and cohosted by Intelligent Enterprise Solution and TATAA Biocenter, highlighted several emerging areas that aim to overcome these limitations. At the symposium, researchers reported new advances in qPCR applications that address stem cell characterization, microRNA profiles, pathogen and biomarker validation, and high-throughput advances for genotyping and gene expression.
Because qPCR assays can detect low amounts of template, several speakers addressed recent findings on genomic expression dynamics at the single-cell level.
One of the presentations at the meeting, “High Throughput PCR and Its Applica-tions,” was delivered by BioTrove’s (www.biotrove.com) CTO, Colin Brenan, Ph.D., just weeks after the company’s OpenArray™ nanoplates were adopted by Applied Biosystems (www.appliedbiosystems.com) for use in end-point PCR applications like SNP genotyping.
Dr. Brenan and four colleagues founded BioTrove in 2000, intent on applying their mechanical engineering skill set in micro- and nanofluidics to the challenge of miniaturizing microtiter plates. The team realized that any system they developed would have to interface with the outside world and that the miniaturized technology had to be easy to use, maintain existing workflow, fulfill expectations of productivity gains, and lower overall costs.
OpenArray, Dr. Brenan noted, provides a high degree of flexibility in designing experiments, allowing multiple samples to be interrogated by multiple PCR tests for a total of 3,072 simultaneous PCR measurements for each microscope slide-sized plate. For example, an OpenArray plate can be loaded such that the expression of 3,072 genes in one sample or 64 genes in 48 samples can be quantitatively measured by real-time PCR.
“This is a real advantage of the OpenArray system over microarrays and the new high-throughput sequencing technologies. How do you scale from 10 or 100 samples to measure gene expression in 1,000 or 10,000 samples?
“To address flexibility issues,” Dr. Brenan added, “we figured out how to accurately and precisely load 33 nanoliter reactions in all 3,072 holes of an OpenArray plate by making the inside of the holes hydrophilic and PCR-friendly and the outside surface of the plate hydrophobic. Primer sets or primer probe sets are dried and locked down inside the holes. In the first heating cycle, primers come off and interact and PCR starts.
“Our product is a consumable, so we had to scale up quickly and with an eye on costs,” Dr. Brenan noted. BioTrove settled on common, inexpensive stainless steel for the nanotiter plates coupled with sophisticated, polymer-coating technology, again using well-proven materials: PEG for the hydrophilic coatings and silanes for the hydrophobic.
The result, according to Dr. Brenan, is a system that can produce a “faster time to answer” compared to the same experiments in microplates with a substantial reduction in reagent costs. “The user gets high-quality information with familiar PCR-based assays that have flexible assay formats for different projects,” Dr. Brenan summarized.
The product has been on the market for about a year and has been used in thermal-cycle endpoint applications such as SNP genotyping, detection of pathogen DNA and RNA target sequences, as well as real-time PCR for quantitative measurement of gene expression. Animal labs use the system as a QC tool, with one customer using a panel of 21 pathogens targeting RNA and DNA run in triplicate.