Microarrays and qPCR
Eppendorf has developed a hybrid technology that combines the major advantages of microarrays (multiplexing and specificity) with real-time PCR (sensitivity and dynamic range). “Current qPCR methods are limited in their multiplexing capabilities by the number of detectable fluorochromes,” reports William Pluester, Ph.D., CEO of Eppendorf Array Technologies. “We have worked to develop a way to combine DNA amplification with array detection (hybridization) in a closed single device, thus eliminating contamination risks while at the same time offering an automated, easy-to-use approach.”
For RAP (real-time array PCR), “a DNA polymerase was genetically engineered for amplification and hybridization to proceed under identical buffer conditions,” Dr. Pluester notes. “In addition, a detection technology was developed to discriminate surface bound fluorescence from unbound label in solution, making buffer changes, washing, or pipetting steps obsolete.”
The company’s first application of RAP will be in rapid detection of nosocomial pathogens and their antimicrobial resistances, a worldwide healthcare issue. “There is a clear unmet clinical need for speed and multiplex data. Results of conventional culture-based tests usually are available too late to impact on clinical decision making.
“Further, selection of such treatment is dependent on accurate identification of the underlying pathogenic causative agents with multiple potential antibiotic resistances. Improper treatment is estimated to result in prolonged hospital stays and in a near doubling of the mortality rates in ventilator-associated pneumonia.”
So far, the company has tested a 31-plex panel for detection of bacteria and their antibiotic resistances in ventilator-associated pneumonia. “We plan to increase this panel to 50-plex in the near future,” Dr. Pluester notes. “We see good correlations between phenotypic data and our significantly faster RAP technology. We just started an extensive program to further validate this application at three university hospitals.”
Other areas in molecular diagnostics that could benefit from a rapid and higher multiplexed assay are genotyping and viral load testing (virology), allele discrimination, SNP detection, and mutation analysis (pharmaco- and functional genomics).
“Furthermore, there are a number of other important applicational areas such as food and feed testing (GMO, pathogens, allergens), veterinary and environmental testing, or forensics, where the promising hybrid approach of RAP could lead to the development of improved rapid multiplexing assays,” he concludes.
The controversy as to whether younger women should or should not have mammograms could be alleviated by a more exact way to determine if and when ductal carcinoma in situ (DCIS) goes malignant. “This is a very troubling issue,” says Ray Mattingly, Ph.D., associate professor, pharmacology, Wayne State University.
“The problem is that mammograms can find DCIS, but in reality, it is only a risk factor, not a death sentence. There are thousands of women who test positive who are only offered the option of watchful waiting. What we want to do is to tell when, if ever, the DCIS will go on to become breast cancer and also offer more targeted treatment.”
Because patient DCIS lesions are tiny they often do not provide enough material to study. Working together with the laboratory of Bonnie F. Sloane, Ph.D., Dr. Mattingly’s group has developed a tractable in vitro model to study DCIS. “This model is based on 3-D overlay cultures in reconstituted basement membranes. We have used them to apply and cross-validate whole-genome microarrays and digital gene-expression analyses (DGE) to characterize genes. We used both methods in three different models to seek common gene signatures for DCIS.”
According to Dr. Mattingly, microarrays are a good place to start such an analysis. “Microarrays can help determine what proteins are being expressed and changed in models. This could provide potential biomarkers as well as treatment targets. Our whole-genome array contained about 22,000 probes. The DGE technology is a generation beyond whole-genome arrays. A microarray will find only what is probed for, whereas DGE takes a sample and sequences everything. This eliminates pre-conceived notions.”
Dr. Mattingly’s group got five million readable sequences using DGE, some of which showed up multiple times, while others were more rare. “The study data was robust. Our bioinformatics collaborators worked to make sense of the data. We found a qualitative concord between the two techniques and came up with a core of 79 genes among all three models.”
The next step is to look for biological patterns and biochemical pathways for the genes already identified. Then they will look for any small molecule inhibitors or collaborate to develop them for new breast cancer therapies.
Nanoscale Protein Arrays
Microarray technologies suffer from several important limitations. Traditional protein-array instrumentation that uses pin-spotting or ink-jetting technology often produces inconsistent features that lead to poor reproducibility and poor accuracy, suggests Jennifer Ohayon, Ph.D., biodiscovery project leader at NanoInk. She says other challenges include low detection limits, the large amount of sample needed for probing microarrays, and slow reaction kinetics.
“These are problems that nanoscale protein arrays can address,” reports Dr. Ohayon. “Moving from the micro- to the nanoscale opens up a whole new way of working with arrays. We have developed several DipPen Nanolithography® nanofabrication technologies with a powerful probe-based deposition technique that uses optimized substrates and next-generation ultrasensitive-detection systems.
“This provides a direct-write spotting technique capable of generating sub-micron-sized features of biomarkers on a solid functionalized glass slide with nearly unlimited real estate. In contrast to typical microarrays that may be 100 microns with donut holes and inconsistent features, our nanoscale arrays are homogeneous with consistent features, providing low coefficient of variations.”
According to Dr. Ohayon, the enhanced biomarker sensitivity of detection afforded by nanotechnology may also provide a more complete understanding of human disease from a systems biology approach because of the ultraminiaturization and sensitivity of the nanoarrays. “Systems biology and personalized medicine go hand in hand. When you look at all components of the system, the use of nanoscale arrays provides a much better snapshot of many of the proteins involved in human disease and response to therapeutics that could ultimately allow one to tailor make a therapeutic.”
The company’s initial studies involved a nanoscale assay for prostate-specific antigen. “We were seeing sensitivities greater than 100 times those seen by other microarrays. This is because the smaller features of nanoarrays tend to focus onto a small spot so analytes can be detected with much greater sensitivity. Later this month, we will be launching our first research kit for the detection of cytokines responsible for human inflammation. An angiogenesis kit, to assess for development of cancer is also currently in development.”
Dr. Ohayon reports that the company is also considering coupling nanoscale arrays with mass spectroscopy in order to develop and interrogate nanoarrays for discovery of new biomarkers. “We fully expect that nanoarrays are an emerging technology that will become more mainstream, especially for the diagnostic kits of the future.”