Nanoliter high throughput quantitative PCR.

Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

A consolidated approach to analyzing data from high-throughput protein microarrays with an application to immune response profiling in humans.

MOTIVATION: DNA microarrays are a well-known and established technology in biological and pharmaceutical research providing a wealth of information essential for understanding biological processes and aiding drug development. Protein microarrays are quickly emerging as a follow-up technology, which will also begin to experience rapid growth as the challenges in protein to spot methodologies are overcome. Like DNA microarrays, their protein counterparts produce large amounts of data that must be suitably analyzed in order to yield meaningful information that should eventually lead to novel drug targets and biomarkers. Although the statistical management of DNA microarray data has been well described, there is no available report that offers a successful consolidated approach to the analysis of high-throughput protein microarray data. We describe the novel application of a statistical methodology to analyze the data from an immune response profiling assay using human protein microarray with over 5000 proteins on each chip.

PhotochemCAD 2: a refined program with accompanying spectral databases for photochemical calculations.

The PhotochemCAD program has been revised extensively. Calculations can be performed using eight modules (oscillator strength, transition dipole moment and natural radiative lifetime, Förster energy transfer, multicomponent analysis, blackbody radiator, artificial spectrum creation, transmission calculation, and analysis of energy transfer among linear multichromophore arrays). The user interface has been streamlined to facilitate visual display, operation of the various modules, input of user data via a wizard and output of spectra and calculations. The database of absorption and fluorescence spectra has been expanded to 150 photochemically relevant compounds. A database of solar spectra has been added. The program runs under Windows and is equipped with extensive literature references and help features, including a tutorial section with video files.

Nanoliter high-throughput RT-qPCR: a statistical analysis and assessment.

Biomarkers discovered from gene expression profiles using hybridization microarrays have made great inroads in the diagnosis and development of safer and efficacious drugs. The candidate gene set is biologically validated by quantitative measurement with reverse transcriptase quantitative PCR (RT-qPCR) and is an effective strategy when implemented with microplates if the number of candidate genes and samples is small. With the trend toward informative candidate gene panels increasing from tens to hundreds of genes and sample cohorts exceeding several hundred, an alternative fluidic approach is needed that preserves the intrinsic analytical precision, large dynamic range, and high sensitivity of RT-qPCR, yet is scalable to high throughputs. We have evaluated the performance of a nanoliter fluidic system that enables up to 3072 nanoliter RT-qPCR assays simultaneously in a high-density array format. We measured the transcription from two different adult human tissues to assess measurement reproducibility across replicates, measurement accuracy, precision, specificity, and sensitivity; determined the false positive rate (FPR) and false negative rate (FNR) of the expressed transcript copies; and determined differences in kinase gene expression reflecting tissue and dosage differences. Using our methodology, we confirm the potential of this technology in advancing pharmaceutical research and development.