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.
Pathway Response Measurements
Proximity ligation assay (PLA), a technique that extends the use of qPCR to the quantitation of proteins, was discussed by David W. Ruff, Ph.D., senior staff scientist at Applied Biosystems. “The central dogma of gene expression involves transcription, RNA processing, and translation, resulting in protein expression.” Presently available real-time PCR assay formats, however, cannot provide insight into the post-translational events that occur in cells.
“The great value of PLA is that it addresses a whole realm of protein biology currently inaccessible to real-time PCR. Soon we will be able to look at pathway responses,” said Dr. Ruff.
PLA requires the binding of two different antibodies to a target antigen, creating a tri-molecular complex. Each of the two antibodies carries a specific oligonucleotide, and this dual entity is called a proximity probe. When in proximity, ligation of the two oligonucleotides will occur in the presence of a connector oligonucleotide and ligase enzyme, generating a full-length PCR template.
Because the technique requires two separate antibody-binding events in close proximity, background signal is minimized. Applied Biosystems currently uses commercially available biotinylated antibodies and oligonucleotide sequences coupled to streptavidin for the construction of the 5´ and 3´ end proximity probe reagents for an off-the-shelf approach.
The technique has shown excellent assay reproducibility in multiday, interinstrument, and reagent batch studies, according to Dr. Ruff. In a dynamic range evaluation of 29 commercially available biotinylated antibodies, 80% performed satisfactorily in the PLA assay without any optimization using recombinant protein antigens, he reported. Excellent performance has also been demonstrated for detection of proteins in cell lysates with targets including cell surface, cytosolic, and nuclear proteins involved in signaling and transcriptional regulation, added Dr. Ruff.
In summary, Dr. Ruff pointed out that PLA is a simple, one-step cell-lysis procedure that requires small amounts of biological sample with an easy workflow, short time to result, and a PCR readout that allows for relative quantitation over a large dynamic range. Because two independent antibody-binding events are required, the amplification signal is specific. Its compatibility with the qPCR-comparative CT method enables reproducible and accurate relative protein quantitation.
In terms of the outlook for the technique, Dr. Ruff foresees applications in pathway responses, and protein modifications as well as correlation of gene- and protein-expression data. Future assay formats will likely accommodate multiplex binding workflows and applications in protein-protein interaction studies.
Roberto A. Macina, Ph.D., director of molecular diagnostics at diaDexus (www.diadexus.com), spoke on “Selection of Prognostic Biomarkers by Quantitative RT-PCR.” diaDexus is a 10-year-old joint venture between SmithKline Beecham (now GlaxoSmithKline), which provided diagnostic rights to certain targets in its pipeline, and Incyte Genomics, which provided diaDexus with access to its genomics database.
In addition to marketing the PLAC® test, reportly the only FDA-approved test for predicting the risk for coronary heart disease and ischemic stroke, diaDexus is developing a diagnostic cancer pipeline including a nucleic acid program that focuses on breast and colorectal cancers, reported Dr. Macina.
He outlined the elaborate process his group uses to hone in on genes that are expressed in specific tissues types. “We focus on mRNA-expression markers,” Dr. Macina told the conference, “and we use numerous selected proprietary markers from our genomics discovery and validation efforts to perform clinical studies in order to apply these markers to unmet medical needs.”
The process involves selecting genes from diaDexus’ pipeline and public databases that are differentially expressed in specific cancers. Public information is then used to determine the function of the protein encoded by each gene and the biological processes in which the protein is involved. Public information also provides clues to which genes to select based on their differential expression in specific cancer clinical applications. In this way, diaDexus includes in its final portfolio genes covering the different biological processes related to the particular clinical application being studied.
Based on the observation that up to 15% of early-stage invasive breast cancer diagnoses currently classified as low risk based on traditional prognostic factors will present with distant recurrence in 10 years, diaDexus began a study to identify a panel of genes that predict outcome in the early-stage, invasive breast cancer low-risk group of LN-, ER+ patients.
A number of clinical parameters were used as factors in the prognosis study, including patient age; tumor size and grade; HER2, ER, and PR status (positive or negative); and finally outcomes, where 29 patients emerged disease free while 30 experience progressive disease. Then, using RT-PCR, diaDexus studied the expression of 32 cancer genes with different biological ontology including differentiation, signal transduction, DNA repair, immune system, cell proliferation, proteolysis, and metastasis.
Finally, using sophisticated mathematical and algorithmic analyses, two of the genes, RAD54L and CCR8, were identified as differentially expressed between the two cohorts and related to survival. In addition to expanding the number of genes tested in this patient population to further assist in the classification of the disease-free and progressive disease populations, Dr. Macina’s group is conducting similar studies in colorectal cancer populations.
Analysis of the miRNA Region
Marc Valer, formerly genomics microfluidics program director at Agilent (www.agilent.com), made a presentation on “Small RNAs in Total RNA: Understanding miRNA Preparations” at the meeting. Subsequently, Peter Barthmaier, business development manager for microfluidics, commented on Valer’s presentation and the capabilities of Agilent’s 2100 bioanalyzer to routinely quantify miRNA in total RNA.
Agilent recently introduced the Small RNA Kit for the 2100, which extends the assay portfolio to the separation and analysis of the miRNA region and other small nucleic acids. The new kit enables the routine quantification and characterization of the miRNA region down to the picogram/ microliter level in any workflow, according to Barthmaier. Coupled with the company’s recent acquisition of Stratagene, the new 2100 bioanalyzer kit is part of Agilent’s strategy to pursue new growth areas while providing customers with more complete workflow solutions.
Polyacrylamide gel electrophoresis (PAGE) is too complex and labor intensive for day-to-day use in miRNA quantification, pointed out Barthmaier. The new small RNA assay removes all previous barriers to routine miRNA sample QC prior to samples being introduced into microarrays and delivers the speed and convenience of an automated system, he added.
“The lab-on-a-chip-based 2100 bioanalyzer platform is ideal for this application because it is far more sensitive than techniques such as slab gels with better resolution and digital output. Like other bioanalyzer kits, automated miRNA analysis of up to 11 samples occurs in just 30 minutes with little human intervention,” Barthmaier explained.
Agilent developed the application for sizing small RNAs from 6 to 150 nucleotides. These small noncoding RNAs are thought to regulate which genes are turned on or off in approximately one-third of all human genes. There are now more than 500 known human miRNAs, with ongoing discovery directed toward an estimated 10,000. MicroRNA expression patterns are associated with a number of tumor types as well as regulating processes such as cellular development, metabolism, and viral infections.
“The enormous potential of this emerging field is driving a great deal of research and highlights the importance of accurate detection of small quantities of miRNA,” Barthmaier said. The Small RNA Kit can detect as little as 50 pg of miRNA out of 10–100 ng of total RNA compared to the typical agarose gel, which might require 20 to 100 times more sample to consistently detect the miRNA fraction, according to Barthmaier. This means researchers can qualify small amounts of precious samples from biopsies, for example, and use only the good samples in more costly miRNA expression-profiling assays.