November 1, 2008 (Vol. 28, No. 19)

High-Throughput Platforms and Real-Time Profiling Advance Discipline

The annual “qPCR Symposium USA” cohosted by Intelligent Enterprise Solutions and the tataa biocenter will be held later this month in California. The symposium will focus on high-throughput platforms, single-cell qPCR, multimarker diagnostics, real-time PCR expression profiling, and data treatment and management.

Several of the presenters previewed their presentations for GEN in this article. Read on for new tips, tricks, and techniques to add to your qPCR tool box.

At Biosearch Technologies, R&D effort is focused on the careful design and delivery of fluorescent-labeled oligos to enable any type of qPCR assay, whether for in vitro diagnostics or genome-wide studies.

Ben Sowers, Ph.D., senior research scientist at Biosearch, is supporting the VelociGene® division of Regeneron, which represents one of two groups developing cell lines with systematic gene knockouts for the NIH-funded Knockout Mouse Project (KOMP). Their contribution will culminate in 3,500 cell lines to cover a large part of the mouse genome.

KOMP Project Process

Dr. Sowers and Biosearch are designing and synthesizing the oligo probes and primers that confirm the success of each gene knockout through qPCR. Two TaqMan® assays target the beginning and end of each gene, an important redundancy since the read-outs from an assay pair can occasionally disagree with one another.

“These are singleplex assays that are essentially one-off reactions, therefore we manufacture the oligos on a small scale,” says Dr. Sowers, “nevertheless, we have to get it right the first time. Given the size of the overall project we lack the luxury of assay optimization by fine-tuning the thermal cycling and reformulating the buffer composition.”

A principal challenge for Biosearch is targeting the primer sets with the proper specificity, to avoid amplifying repetitive sequences and gene homologs. If left unaccounted for, nonspecific amplifications would be the top reason for an assay to fail. Therefore, Dr. Sowers queries NCBI databases using electronic PCR to define unique amplicons through a fuzzy matching strategy.

From an administrative perspective, such a large number of assays can be difficult to design and track, so batch processing becomes essential. RealTimeDesign is a software program that was purpose-built for high-volume sequence design by Celadon Laboratories. It accommodates up to 10 different targets to process en masse, and after design the sequences are stored in the software’s archives.

This centralized database helps track the progress of this project and review assays at a later time. According to Dr. Sowers, applying this program requires no IT oversight because it operates entirely through a web browser; there is nothing to install and maintain.

To date, Biosearch has completed approximately 50% of the 7,000 qPCR assays for the KOMP project. Through the use of RealTimeDesign it has kept the inherent failure rate down to 8%, with success defined as a positive amplification within two cycles of when it’s expected. For detection, Dr. Sowers relies on Biosearch’s Black Hole Quencher® (BHQ®) probes to identify each amplicon, and occasionally BHQplus™ probes to provide a clean result for regions that are AT-rich.

Biosearch supports the KOMP project with its high-throughput design software and oligo manufacturing. This experience highlights the need for a couple of key features to improve upon the functionality of RealTimeDesign. Dr. Sowers has also gathered important suggestions from the research community. All of this feedback will culminate in a new version of the software to launch before November. Biosearch makes RealTimeDesign available at no charge.

LNA Probes

Alexander Aristarkhov, Ph.D., director of science and technology at Exiqon, will be speaking at the conference about the company’s ongoing effort to amplify and detect miRNA expression levels. These short, noncoding RNA molecules modulate gene expression by base-pairing mostly with the 3´ untranslated region of their target mRNA. This likely affects many aspects of the posttranscriptional life of those targets.

Those principles of regulations were completely overlooked. What really provides Exiqon a competitive advantage is its exclusive rights for the use of Locked Nucleic Acid (LNA™) probes.

The unprecedented challenge for scientists in this area is reliable detection of RNA this size. miRNAs are small, about 21 nucleotides, not much different than the size of most PCR primers (20–22 nucleotides).

To capture these small molecules, Exiqon made use of its LNA-based primer design. Exiqon combines the use of LNA probes with SYBERgreen detection methods to generate and detect a specific signal to track the amplification reactions of these targets. LNA allows shorter probes with exceptional affinity and mismatch recognition in comparison to DNA-based probes, Dr. Aristarkhov notes.

It is the biology of these small RNAs that makes them interesting to study. The heterogeneity of the population for many miRNAs is well documented. The miRNAs, which can differ in length, show a definite lack of precision at both ends. What has yet to be determined is whether this lack of precision at the ends is due to differential degradation or perturbation during synthesis. Or, is it merely an artifact of isolation protocols?

“We’ve developed a microarray platform technology with validated assays for miRNAs in the cell requiring as little as 30 ng of total RNA,” says Dr. Aristarkhov, “these validated assays allow us to overcome the challenge of measuring heterogeneous miRNA amplification products. Not only are these miRNA targets small, but they can also differ at only one or two nucleotides at any position along the length of the miRNA.”

“While this provides a unique signature to track the cell physiology, it is challenging to capture and record the levels appropriately. We are focusing on miRNAs and paying close attention to all aspects of biology and detection of small regulatory RNAs to generate accurate and reliable data.”

The ultimate goal for Exiqon is to develop a complete portfolio of technological platforms based on LNA for detection of miRNAs using microarray, qPCR, in situ hybridization, and gene knockdowns. It will guide researchers from miRNA discovery to determination of functions.

Real-Time Application Kits

At Invitrogen, the R&D effort in qPCR research is to generate optimized, real-time PCR kits that work on all brands and types of real-time PCR instruments and cover applications such as gene-expression profiling, genotyping, high-resolution melting curve (HRM), and analysis and multiplexing.

A tall order, but Gothami Padmabandu, Ph.D., the qPCR R&D lead, says the company has pulled it off. The next generation of Express qPCR and qRT-PCR kits have been formulated to support both standard (40 cycles in 120 min) and fast (40 cycles in 40 min) cycling protocols. The key for the latter methodology is to overcome the loss of sensitivity and increased variability that are often seen during fast reactions run for high-throughput analysis.

An additional performance benefit of the new kits is the inclusion of a heat-labile uracil DNA glycosylase (UDG) enzyme to prevent carryover contamination in all reactions, including one-step qRT-PCR.

Whereas previous generations of reagents could only include UDG protection in two-step formats due to the thermal stability of most UDG enzymes, the combination of increased thermostability of Invitrogen’s SuperScript®III Reverse Transcriptase and heat-labile UDG makes it possible to incorporate for the first time UDG in one-step reactions, according to Dr. Padmabandu.

EXPRESS Two-Step qRT-PCR kits also contain the new SuperScript® VILO™ cDNA Synthesis Kit, which is SuperScript III Reverse Transcriptase formulated in an enhanced buffer system to provide higher cDNA yields.

A final feature benefit in the new Express kits designed for SYBR detection is the inclusion of the SYBR®GreenER™ dye. The kits are designed to minimize nonspecific amplification and enable the detection of even low copy targets with a dye that is brighter and less PCR inhibitory. The kits can be used for emerging technologies such as HRM for gene-scanning methylation status analyses or detection of SNPs.

Miniaturizing Reactions

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.

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