Hot New Applications
Mirus Bio offers an extensive line of products for the investigation of microRNAs (miRNAs). These are small, noncoding RNAs of 19 to 24 nucleotides involved in regulation of messenger RNA.
MicroRNAs are members of a class of small regulatory RNAs including small interfering RNAs (siRNAs) that control the expression of downstream gene targets, including transcription factors, oncogenes, and tumor suppressor genes. Because of their association with cancer development, diagnosis, and prognosis, it is believed that their signatures can be used to detect and classify cancers and predict the severity of disease.
Technological advances by Mirus and other companies in expression profiling and in the ability to collect minute quantities of tissues allow a growing number of global transcriptional studies. Microarray technology is now well established for miRNA studies.
According to Shannon Bruse, Ph.D., director of scientific operations at Mirus, “MicroRNA expression-profiling analysis has become a prominent application of microarray technology, measuring the presence and relative amount of specific miRNAs.” In order to quantify the fluorescent signal from the hybridization step, the Mirus Bio’s Label IT platform covalently attaches label to nucleic acids. “Samples labeled directly with the Label IT reagents do not require enzymatic replication, while at the same time they yield sensitive hybridizations,” Dr. Bruse adds.
Taking the RAP
“Rapid pathogen testing is an unmet clinical need that PCR combined with microarrays can fill,” says Christof Henne, Ph.D., key account manager for diagnostics at Eppendorf Diagnostics. To address this void, the company has developed the RAP™ (Real-Time Array PCR) that it believes combines the advantages of qPCR and those of microarrays.
According to Dr. Henne, RAP technology brings together the sensitivity and reproducibility of PCR with the rapid multiplexing capability of microarrays.
A critical feature in the construction of the Eppendorf platform was the genetic engineering of the active site of the polymerase enzyme so it would function at high salt concentrations. This allows stable surface binding under conditions that would ordinarily be nonpermissible. Microarrays do not lend themselves to quantification, and they have a low dynamic range, but these drawbacks can be overcome when they are combined with effective PCR.
Dr. Henne mentions that the Eppendorf platform can be coupled to commercial systems for PCR-based DNA testing. “This is a niche market that is rapidly expanding with an increasing number of assays,” he notes. One example of an integrated commercial system for PCR-based DNA testing is designed by Cepheid, combining sample preparation, DNA amplification, and detection. “The system was originally developed for anthrax testing, but the company is now expanding its diagnostic offerings,” Dr. Henne comments.
“For a point-of-care test for infectious disease diagnosis to be acceptable, it must have robustness and it must have built-in controls that inform the user of his mistakes, with clear positive and negative controls,” he continues. “The more automated the platform is, the better for the patient, so the only remaining variable is the sample quality.”
Personalized medicine is a rapidly developing area to which the RAP technology lends itself, Dr. Henne notes. Voluminous information is generated per patient, and it will be necessary to describe the patient in much more individualized terms, taking into account genetic subtypes that describe sensitivity to cancer and other disease processes.
“The combination of multiplexing ability with rapid testing will be essential in building of personalized medicine platforms,” he concludes.
An Array of Possibilities
Since array technology came on the scene in the early 1990s, it has experienced a number of advances that have greatly expanded its range of possibilities, and it is widely used today for gene-profiling studies that may produce drug targets or potential biomarkers. Yet doubts still exist concerning the reproducibility and compatibility of array data generated in different laboratories and on different platforms. Until these technical and experimental issues can be resolved, the technology will not reach its full potential as a tool for drug discovery.