Oligonucleotide-and cDNA-based microarrays comprising a subset of Neisseria meningitidis genes were assessed for study of the meningococcal heat shock response and found to be highly suitable for transcriptional profiling of N. meningitidis. Employing oligonucleotide arrays encompassing the entire genome of N. meningitidis, we analyzed the meningococcal heat shock response on a global scale and identified 55 heat shockderegulated open reading frames (34 induced and 21 repressed).Sequencing of the genomes of Neisseria meningitidis serogroup A and serogroup B strains provided us with a tremendously broad range of information (15,23). The next step is the elucidation of gene expression patterns and gene product function on a genome-wide scale. DNA microarrays offer an ideal tool for high-throughput investigation of gene regulation on the transcriptional level (for review, see references 4, 13, and 17). The two most commonly employed DNA microarray platforms are oligonucleotide and cDNA arrays. Here we performed a comparative analysis of the suitability of both technology platforms for transcriptional profiling of N. meningitidis.The two main features of DNA microarray performance are sensitivity (signal intensity) and specificity (ratio of specific to nonspecific hybridization). Additional care must be taken to standardize experimental conditions and to avoid the detection of false-positive signals (12). In order to validate gene expression modulations of N. meningitidis observed using cDNAbased and oligonucleotide-based microarrays, we first performed parallel hybridizations of identical RNA samples to the same slide. Microarrays containing probes specific for 60 genes selected from the published genome sequence of N. meningitidis serogroup B strain MC58 (23) were produced (Table 1). For cDNA-arrays, internal fragments of each open reading frame (ORF) (300 to 560 bp) were PCR amplified. For oligonucleotide arrays, oligonucleotides (40-mers, three per gene) comprising gene-specific internal fragments (covering 5Ј, central, and 3Ј parts) were designed. All oligonucleotides (manufactured by MWG-Biotech AG, Ebersberg, Germany) carried a C6 amino linker modification at the 5Ј end for covalent attachment to the slide surface. Each probe was spotted 5 (oligonucleotides) or 10 times (PCR products) per array using the Affymetrix 417 Arrayer (MWG-Biotech AG). PCR products were spotted on CMT-GAPS-Coated Slides (Corning, Wiesbaden, Germany), oligonucleotides were spotted on Super Aldehyde Slides (TeleChem International, Sunnyvale, Calif.), and the slides were processed according to the manufacturers' instructions.Cultures of N. meningitidis strain MC58 (24) were grown to mid-logarithmic growth phase (optical density at 600 nm [OD 600 ] ϭ 0.5/5 ϫ 10 8 CFU/ml) at 37°C in supplemented proteose peptone medium and RNA isolated as previously described (5). The RNA was split into two aliquots, and onehalf was labeled with Cy3-dCTP, the other with Cy5-dCTP (Amersham Pharmacia, Freiburg, Germany) during a firststrand reverse transcri...