Escherichia coli Spotted Double-Strand DNA Microarrays: RNA Extraction, Labeling, Hybridization, Quality Control, and Data Management

Highly parallel hybridization of nucleic acids on glass slides has successfully been applied to measure RNA and DNA abundances in Escherichia coli (1 –4 ). In this chapter, we summarize our experience in working with E. coli DNA microarrays accumulated over a 4-yr period. Typically, we printed and used E. coli DNA microarrays containing roughly 6000 spotted elements. These included 4200 amplicons of E. coli open reading frames (ORFs), 112 amplicons of genes encoding stable RNAs, and more than 1500 control elements and replicates. We describe the methods for total RNA extraction, mRNA enrichment of total RNA, cDNA labeling via direct and indirect incorporation of fluorophors, and microarray hybridization. Additionally, we present strategies for optimizing microarray hybridizations and descriptions of several Internet-based tools useful in analyzing data from array experiments.