Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury.

Background: MicroRNAs (miRNAs) are endogenous, small noncoding RNAs. Because of their size, abundance, tissue specificity, and relative stability in plasma, miRNAs hold promise as unique accessible biomarkers to monitor tissue injury.

Methods: We investigated the use of liver-, muscle- and brain-specific miRNAs as circulating biomarkers of tissue injury.

Development of a microarray platform for FFPET profiling: application to the classification of human tumors.

Background: mRNA profiling has become an important tool for developing and validating prognostic assays predictive of disease treatment response and outcome. Archives of annotated formalin-fixed paraffin-embedded tissues (FFPET) are available as a potential source for retrospective studies. Methods are needed to profile these FFPET samples that are linked to clinical outcomes to generate hypotheses that could lead to classifiers for clinical applications.

Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter Assay System.

Background: We assessed NanoString's nCounter Analysis System for its ability to quantify gene expression of forty-eight genes in a single reaction with 100 ng of total RNA or an equivalent amount of tissue lysate. In the nCounter System, multiplexed gene expression target levels are directly detected, without enzymatic reactions, via two sequence-specific probes. The individual mRNA is captured with one mRNA target sequence-specific capture probe that is used in a post-hybridization affinity purification procedure.

Improved high-throughput ultrafiltration process enables cRNA purification for gene expression profiling.

Ultrafiltration of nucleic acids has been used for a wide variety of applications, including sequence reaction purification and amplicon cleanup prior to spotting onto microarrays. Here we describe a novel process, using ultrafiltration, that purifies cRNA products for sensitive downstream applications. Initial attempts at this high-throughput purification for cRNA resulted in low sensitivity when compared against an industry standard (silica-based bind, wash, and elute purification).

Characterization of globin RNA interference in gene expression profiling of whole-blood samples.

Background: Blood-based biomarker discovery with gene expression profiling has been hampered by interference from endogenous, highly abundant alpha- and beta-globin transcripts. We describe a means to quantify the interference of globin transcripts on profiling and the effectiveness of globin transcript mitigation by (a) defining and characterizing globin interference, (b) reproducing globin interference with synthetic transcripts, and (c) using ROC curves to measure sensitivity and specificity for a protocol for removing alpha- and beta-globin transcripts.

Methods: We collected blood at 2 sites and extracted total RNA in PreAnalytiX PAXgene tubes.

Reagent preparation and storage for amplification of microarray hybridization targets with a fully automated system.

The advent of automated systems for gene expression profiling has accentuated the need for the development of convenient and cost-effective methods for reagent preparation. We have developed a method for the preparation and storage of pre-aliquoted cocktail plates that contain all reagents required for amplification of nucleic acid by reverse transcription and in vitro transcription reactions. Plates can be stored at -80 degrees C for at least 1 month and kept in a hotel at 4 degrees C for at least 24 h prior to use.

Use of a mixed tissue RNA design for performance assessments on multiple microarray formats.

The comparability and reliability of data generated using microarray technology would be enhanced by use of a common set of standards that allow accuracy, reproducibility and dynamic range assessments on multiple formats. We designed and tested a complex biological reagent for performance measurements on three commercial oligonucleotide array formats that differ in probe design and signal measurement methodology. The reagent is a set of two mixtures with different proportions of RNA for each of four rat tissues (brain, liver, kidney and testes).

A microarray platform comparison for neuroscience applications.

To address the need for high sensitivity in gene expression profiling of small neural tissue samples ( approximately 100 ng total RNA), we compared a novel RT-PCR-IVT protocol using fluor-reverse pairs on inkjet oligonucleotide microarrays and an RT-IVT protocol using 33P labeling on nylon cDNA arrays. The comparison protocol was designed to evaluate these systems for sensitivity, specificity, reproducibility, and linearity. We developed parameters, thresholds, and testing conditions that could be used to differentiate various systems that spanned detection chemistry and instrumentation; probe number and selection criteria; and sample processing protocols.

Effects of atmospheric ozone on microarray data quality.

A data anomaly was observed that affected the uniformity and reproducibility of fluorescent signal across DNA microarrays. Results from experimental sets designed to identify potential causes (from microarray production to array scanning) indicated that the anomaly was linked to a batch process; further work allowed us to localize the effect to the posthybridization array stringency washes. Ozone levels were monitored and highly correlated with the batch effect.

Functional characterization of a conducting polymer-based immunoassay system.

Experiments have been performed to characterize the electrical properties and functionality of a poly(3-hexylthiophene)-coated platinum electrode developed as a sensor for immunoassay read-out. Admittance measurements were performed on the coated electrodes as a function of frequency. The admittance spectra obtained show that the sensor is capacitive in nature.

Measurement of acetylcholine receptor function in microcircuit-coupled myoblasts.

An electrophysiological measurement principle for long-term, noninvasive monitoring of the nicotinic acetylcholine receptor (nAChR) function is described. The measurement is based on the ability to record agonist-induced depolarizations of clonal myoblasts that have formed high impedance seals with extracellular microcircuit electrodes. The technique appears promising for several types of assays and environmental monitoring applications.

Immobilization of flavoproteins on silicon: effect of cross-linker chain length on enzyme activity.

The effect of cross-linker chain length on the activities of choline oxidase (ChO) and glucose oxidase (GOx) immobilized on oxidized silicon wafers has been investigated for the cross-linkers N-succinimidyl 4-maleimido-butyrate (GMBS) and N-succinimidyl 6-maleimidocaproate (EMCS). Enzyme activities were determined with an indirect fluorometric assay based on the production of hydrogen peroxide. Immobilization of ChO or GOx onto oxidized silicon with either cross-linker resulted in an 86-99% loss in enzymatic activity relative to the soluble form of the flavoprotein.

Deep UV photochemistry of chemisorbed monolayers: patterned coplanar molecular assemblies.

Deep ultraviolet (UV) irradiation is shown to modify organosilane self-assembled monolayer (SAM) films by a photocleavage mechanism, which renders the surface amenable to further SAM modification. Patterned UV exposure creates alternating regions of intact SAM film and hydrophilic, reactive sites. The exposed regions can undergo a second chemisorption reaction to produce an assembly of SAMs in the same molecular plane with similar substrate attachment chemistry.

Modulation of a gated ion channel admittance in lipid bilayer membranes.

A future class of amperometric biosensors may utilize gated ion channels such as acetylcholine and glutamate receptors as chemical detection components. In this study, bilayer lipid membranes containing voltage-dependent anion channels (VDAC) were used to model an ion-channel-based biosensor which could continuously monitor AC amperometric changes resulting from induced changes in channel conductance. The in-phase and quadrature components of the induced alternating membrane current were monitored as a function of the applied DC offset voltage which was superimposed on the sinusoidal test voltage.

Fabrication of key components of a receptor-based biosensor.

A three-pronged approach was taken to the development of receptor-based bisensors. First, asymmetric bilayer membranes were developed with one monolayer adaptable to the particular receptor of interest and the other monolayer polymerized to enhance membrane stability. Second, alamethicin and calcium channel complexes were introduced into the stabilized membrane and tested for ion-channel function.