Linear RNA Amplification and Microarray Hybridizations Total RNA was isolated from a human breast tumor, a human lung squamous cell carcinoma, and from two cultured breast tumor epithelial cell lines

DNA microarrays are a powerful tool for functional genomic studies. For many microarrays, pieces of DNA are deposited onto known locations on specially coated glass slides, and then these arrays are hybridized with fluorescently labeled cDNA molecules (1). One important aspect of all microarray studies is the replication of data; for the microarray community, sample replicates are of the utmost importance, and the more replicates one performs, the more statistical power can be gained (2). The expense of performing replicate microarray experiments, however, greatly adds to the overall cost and, in many cases, is impractical due to economic constraints. Typically, a single DNA microarray is used only once; however, there are several characteristics of long oligonucleotide arrays (3) that, when coupled with a linear amplification sample labeling protocol that produces cRNA, makes them a candidate for alkaline-based stripping techniques (i.e., removing the labeled probe from the microarray for reuse, as is done with Southern blot analysis). First, the DNA oligonucleotides are covalently bound to the coated surface of the glass slide. Second, the glass slide and its coating are minimally damaged by base with limited treatment times. Third, the arrays can be utilized with a linear amplification protocol that produces fluorescent cRNA, not cDNA, for hybridization. Microarrays hybridized using this protocol can be stripped by taking advantage of the selective degradation of RNA under alkaline conditions while the DNA (target probes) remains intact. Sodium hydroxide has been used to degrade RNA during the extraction of DNA from biological materials for many years. It is also used to destroy input RNA in a variety of fluorescent dye cDNAlabeling methods for microarray applications (4). However, most “stripping” protocols typically use temperatures around 95°C (5–7), which is above the melting point of hybridized cRNA-DNA complexes but is high enough to damage the slide coating. Here we describe a methodology for stripping some DNA microarrays that can then be reused with a high level of reproducibility.