High Precision RPPA: Concept, Features, and Application Performance of the Integrated Zeptosens Platform.

An integrated reverse phase protein array (RPPA) platform shall allow the precise monitoring of expression level and changes of proteins and their functional states in a highly parallel manner even when samples exhibit a complex matrix like in tumor tissues and are available only in very limited amounts. Ideally the full workflow from sample preparation to data visualization shall be covered.This book chapter describes the key elements of the integrated Zeptosens RPPA platform. It addresses critical platform and process design requirements, considerations, and elements as well as critical process steps and quality aspects. Sophisticated instrumentation, high sensitivity readout, and dedicated chip and assay handling equipment act in concert with streamlined protocols, optimal reagents, and dedicated lab equipment in the hands of trained users to achieve an outstanding overall performance of the realized system. Based on results from comprehensive signaling protein and pathway profiling studies targeted for preclinical drug efficacy testing and development, it gives an overview of application performance by means of coefficients of variation (CVs) that can be achieved for assay signals from technical and biological sample replicates with this state-of-the-art integrated RPPA platform and process.The Zeptosens RPPA platform has proven to provide valuable biological information with a high level of confidence and has shown its validity in generating sound mechanistic as well as prognostic and predictive information when analyzing cell and tissue materials on the functional protein level.

Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription.