Sunday, January 1, 2012

RNA Reference Materials for Gene Expression Studies

This flowering of ingenuity is acceptable for hunting candidate genes with one format and verifying results by more common techniques, but for clinical applications, this variety is an obstacle. The typical application envisions a multiparametric “gene-expression signature” in which the expression patterns for many genes are combined to generate a “classifier” for diagnosis or prognosis. Laboratory 1, which uses array system brand A, publishes a well-designed study showing that a gene-expression signature distinguishes benign from malignant omphalomas. How should Laboratory 2, which uses brand X, adapt the “signature”? Clinical studies are also hampered by lack of well-defined controls. This problem is analogous to deciding, without benefit of reference materials, which of two immunoassays is better—when they use independently derived antibodies and different calibrators and controls—and then making this decision for 10 000 immunoassays at once. Some suppliers already provide tools to control for variation, including replicate probes, “spike-in” RNA controls, normalization algorithms, and image-quality metrics, but these also differ among formats. For comparing results among methods, it would be decidedly helpful to have widely available, standardized, renewable pools of RNA species that could monitor RNA purification, monitor cDNA labeling, verify sensitivity, and serve as controls.

During an actual assay, the APS could be cohybridized with a complex sample in two-color format. In the single-color format, the APS and the complex sample would have to be hybridized to separate arrays. The performance of a simple cRNA pool will not necessarily reflect performance of an array system with a more complex mixture. For such a format perhaps a complex sample could be labeled in parallel with and without addition of an APS, and then hybridized to separate arrays (recovery experiment).

The UHS (“spike-in” controls) would provide information on efficiency of cDNA synthesis/labeling, uniformity of hybridization, and sensitivity of detection. A pool added to samples before RNA purification could monitor that process. The utility of APS and UHS for QPCR is clear. The proposed number of materials exceeds needs but will not resolve the fundamental question of how best to normalize QPCR.
Posted by at


Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1-4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Currente Sensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.

Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5,6] and viruses [7,8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.

Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens.