BioTrove CTO and cofounder Colin Brenan, Ph.D., explained how the company has overcome the macro-to-micro barrier in the miniaturization of qPCR reactions by developing the OpenArray™ platform, which supports high-speed nanoliter PCR for simplified multianalyte genetic testing.
BioTrove has already formed a partnership with Applied Biosystems (ABI) to commercialize the OpenArray platform for SNP genotyping applications with ABI’s TaqMan SNP Genotyping Assays. At the conference, BioTrove will display an OpenArray system for qPCR reactions that can be performed in 33 nL total reaction volumes under standard cycling conditions, says Dr. Brenan.
“The OpenArray slide is a thin metal slide with finely machined holes that serve as wells for the PCR reaction,” he adds. “We confine the aqueous reactions within the wells by first creating a hydrophilic and PCR-friendly environment in each well surrounded by a hydrophobic environment above and below the wells. In this way all the components for the reaction can be added to and contained within the wells by simple capillary action from standard pipette tips.”
Each slide can hold 3,072 SYBR Green qPCR assays, which can be divided into a number of different sample-assay configurations. One common configuration is 48 samples with 64 independent reactions per sample, while others are 128 reactions against 24 samples and 256 reactions against 12 samples. At 3,072 qPCR reactions, that is the equivalent of eight 384-well plates on a single slide.
Imagine the savings in labor, sample, and reagents over the cost of microplates and microarrays. Each reaction is run as a singleplex, which reduces the complexity and avoids the need for exhaustive reaction condition optimization, such as that needed for development of multiplexed PCR reactions.
BioTrove supplies either custom OpenArray slides preloaded and quality-checked with customer primers, or OpenArray slides with panels of prevalidated primers targeting genes involved in human disease or biological pathways of interest in disease research.
This new product line of prevalidated qPCR assay panels, called OpenArray Pathways, allows quantitative measurement of gene expression in up to four samples by panels of over 600 human genes covering ADMETox, apoptosis, cancer, cardiovascular disease, inflammation, the entire kinome and the major signal transduction pathways, plus 12 housekeeping genes as endogenous controls in quadruplicate.
BioTrove provides the end-user of the OpenArray system preloaded OpenArray plates, the thermal cycling cassette and fluids, plus the data analysis CD for the assay. The instrumentation required to run the system includes the OpenArray AutoLoader to load samples and the NT Cycler™/NT Imager™.
This robust platform enables the processing of >18,000 qPCR analyses by one technician in a single day, according to Dr. Brenan.
BioTrove is seeing market growth in all major segments—from discovery (9% growth), where customers are processing few samples to look at large numbers of genetic markers, to validation (11% growth), where customers are processing more samples to look at a subset of markers, to screening (18% growth), where customers process large numbers of samples to look at a small set of markers.
This trend sets the stage for the move of this platform into molecular diagnostics, which is being explored through partnership in the areas of pathogen testing, blood banking, and translational medicine. Dr. Brenan will be sharing case studies of each of these in his presentation at the conference.
Jinping Yang, Ph.D., senior director of manufacturing at SABiosciences, says the company is best known for its RT2 Profiler PCR Arrays, a portfolio of products that enable customers to analyze gene-expression profiles of gene panels in signal transduction or disease-related pathways using qPCR.
In support of the PCR Array portfolio, Dr. Yang and her team have been working to optimize the company’s data-analysis software package. This easy-to-use package is now available as a fully integrated web-based tool that automatically performs DCt-based fold change calculations from uploaded raw cycle data.
“The web portal delivers results not only in a tabular format but also in scatter, volcano, cluster-gram, and multigroup plots,” says Dr. Yang. “We enable the researcher to perform any pair-wise comparison between groups of experimental replicates they want. It is easy to make side-by-side comparisons. We are considering adding functionality in the PCR array data-analysis software to present the effects of PCR reaction efficiencies in the final data analysis.”
Dr Yang will show symposium attendees how PCR amplification efficiency impacts the interpretation of qPCR results. The plug-and-play data-analysis software is freely available on the web. The analysis package is designed to accommodate raw data input from any qPCR experiment.
Beyond the effort in data analysis for qPCR, SABiosciences is developing new RT-PCR arrays. The company recently launched a Chromatin immunoprecipitation kit (ChIP) that includes a soup-to-nuts solution for capturing and analyzing non-ORF of the genome. SABiosciences will also soon be launching products for profiling DNA methylation associated with human diseases and stem-cell growth and differentiation.