9:45 Investigating microRNAs by PolyA Tailing-B-based RT-PCR
Rui Shi, Ph.D., Research Associate, Forestry and Environmental Resources, North Carolina State University
MicroRNAs (miRNAs) are short endogenous non-coding RNAs involved in a remarkable range of biological pathways in plants and animals. For better understanding and applying the knowledge of miRNAs, it is important to develop simple and efficient methods for monitoring miRNA expression. Here we will introduce the designing of a polyA tailing based RT-PCR for miRNA analysis, and our efforts in optimization and modification of this approach.

10:15 Comparison of RT-PCR, NASBA and Real-Time (RT)-PCR for the Detection of Noroviruses
Alain Houde, Ph.D., Research Scientist, Department of Food Safety and Quality, Agriculture and Agri-Food - Canada
Different norovirus (NoV) molecular detection systems were evaluated and compared in parallel for their performance and analytical sensitivity in clinical stool samples. The same primer and probe sets were used for each assay on the same extracted or diluted RNA samples. Using non diluted fecal specimens, RT-PCR, NASBA and real-time TaqMan RT-PCR methods were found equally suitable for NoV detection, but the NASBA assay showed some reproducibility discrepancies. No difference was observed in RT-PCR results obtained from dot blot and gel visualization. The NASBA method was more sensitive, by at least 2 logs, than conventional RT-PCR but showed similar limits of detection as the TaqMan RT-PCR technique. The TaqMan RT-PCR assay was reliable with a high analytical sensitivity and has shown the capability of detecting one genomic equivalent copy. In a clinical context, RT-PCR, NASBA and real-time TaqMan RT-PCR methods using undiluted samples were all suitable for the detection of NoV, however the NASBA assay provided less consistent signals.

11:15 Novel ZnO Nanomaterial Platforms in Enhanced Fluorescence Detection
Jong-in Hahm, Ph.D., Assistant Professor, Chemical Engineering, The Pennsylvania State University
This talk presents an overview of our on-going nanomaterials research, aiming to provide more rapid, sensitive, and accurate detection of genetic and protein markers. Specifically, this talk will focus on the remarkably enhanced optical detection of DNA and proteins which is enabled by the use of nanoscale zinc oxide (ZnO) platforms. Fluorescence detection is currently one of the most widely used methods in the areas of basic biological research, biotechnology, cellular imaging, medical testing, and drug discovery. Using model protein and nucleic acid systems, we demonstrate that engineered nanoscale ZnO nanostructures can significantly enhance the detection capability of biomolecular fluorescence. Without any chemical or biological amplification processes, nanoscale ZnO platforms enabled increased fluorescence detection of these biomolecules when compared to other commonly used substrates such as glass, quartz, polymer, and silicon. This ultrasensitive detection was due to the presence of ZnO nanomaterials, which contributed greatly to the increased signal to noise ratio of biomolecular fluorescence. We also demonstrate the easy integration potential of ZnO nanostructures into periodically patterned platforms which, in turn, will promote the assembly and fabrication of these materials into multiplexed, high-throughput, optical sensor arrays. These ZnO platforms will be extremely beneficial in accomplishing highly sensitive and specific detection of biological samples involving nucleic acids, proteins and cells, particularly under detection environments involving extremely small sample volumes of ultratrace-level concentrations.

11:45 Reagent Qualification and its Impact on Nucleic Acid Detection
Uplaksh Kumar, Ph.D., Senior Manager, Manufacturing, Digene Corp.
Well qualified reagents are critical to the performance of any assay. As assays for analyte detection are becoming more sensitive and instrument dependent it is important to have a reagent set that is qualified and stable. Reagent formulations that include complex nucleic acids molecules (DNA and RNA) have to be well qualified to insure that results and claims made by the assay are authentic and reproducible. By putting the rights controls in place one can insure that data from the assay is sample dependent and not dependent on reagent variability.

2:15 Predictive Modeling of Base Call Resolution on Re-Sequencing Microarrays: Design Used for Pathogen Detection
Anthony Malanoski, Ph.D., Chemical Engineer, U.S. Naval Research Laboratory
For diagnostic organism detection, all nucleic acid-based detection methods face requirements to choose regions that provide complete coverage of all possible variants of a target while not responding to non-target material. Primer design and amplification techniques are integral to meeting these requirements although what role they play differs depending on the oratory detection method (false positive and negative are inevitable sometime). Our group has adapted re-sequencing microarray technology and demonstrated their great potential for simultaneously detecting bacterial and viral pathogens, and virulence markers Our initial work has caused us to understand that the capabilities and requirements of probes for this method differ markedly from other microarray based systems. In order to extend our abilities to design new microarrays for new organisms and better understand the capabilities of this platform, we have developed a model that accurately predicts the result of the interaction of any fragment of DNA and a re-sequencing microarray probe. Improved understanding of the capabilities of the re-sequencing array has implications to other aspects of the diagnostic method such as altering primer selection criteria to enhance the performance of the assay. This model now allows us to develop computational algorithms to be applied in future designs so that the best probes requiring the minimal amount of microarray space can be selected.

5:15 DNA Biochips, microPCR, and Hand-Held Gene Analyzers:
The State of the Science
Syed Hashsham, Ph.D., Edwin Willits Associate Professor, Civil and Environmental Engineering, Michigan State University
DNA biochips are extremely powerful for detecting many gene targets in parallel but are not as sensitive as real time PCR. Both DNA biochips and PCR are laboratory-based assays requiring expensive equipment and skilled personnel. Micro-PCR or on-chip PCR is a recent development targeted at miniaturizing the PCR and real time PCR assay for hand-held and point of care devices. Combining DNA biochips and micro-PCR is the logical next step but it requires integration of multiple miniaturized components for the purpose of sample processing, amplification, and detection. This presentation will review the state of science in the area of DNA biochips and micro-PCR, highlight some of the requirements of a hand-held device that may house these components, and share some of the results associated with each.