title: A high-density quantitative nuclease protection microarray platform for high throughput analysis of gene expression.
abstract: The quantitative nuclease protection assay (qNPA) is a very simple and highly sensitive method for measuring mRNA transcripts, can be used on a variety of sample types, and is amenable to high-throughput sample processing. We have combined the power of the qNPA assay with the density of a DNA microarray to create a qNPA Microarray platform. This platform is compatible with common laboratory equipment: it uses fluorescence-based detection, can be analyzed with common microarray scanners, and is in an SBS footprint with 96-well layout for high-throughput applications. Here, we demonstrate the characteristics of a qNPA Microarray slide that contains up to 1700 gene elements per well. We show that the new platform can reliably detect transcripts at levels as low as 10fM with median CVs below 12%. On a standardized set of samples, the qNPA Microarray detected the same trends in gene expression as the original qNPA technology, real time qPCR, and Affymetrix GeneChip DNA Microarrays. Given its ease of use, compatibility with multiple sample types, high-throughput capabilities, and its integration with standard laboratory equipment, the qNPA Microarray is a powerful new platform for gene expression research.
authors.affiliation: BIO5 Institute, University of Arizona, Tucson, United States.
authors.forename: K M
authors.initials: KM
authors.lastname: Bourzac
authors.forename: M P
authors.initials: MP
authors.lastname: Rounseville
authors.forename: X
authors.initials: X
authors.lastname: Zarate
authors.forename: V S R K
authors.initials: VS
authors.lastname: Maddula
authors.forename: D C
authors.initials: DC
authors.lastname: Henderson
authors.forename: J A
authors.initials: JA
authors.lastname: Luckey
authors.forename: B
authors.initials: B
authors.lastname: Seligmann
authors.forename: D W
authors.initials: DW
authors.lastname: Galbraith
mesh_terms: D002460:Cell Line; D015342:DNA Probes / Q000378:metabolism; D030541:Databases, Genetic; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D023282:Nuclease Protection Assays / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D015342:DNA Probes