Design, optimization and validation of genomic DNA microarrays for examining theClostridium acetobutylicum transcriptome

• Published in: Biotechnology and bioprocess engineering Vol. 10; no. 5; pp. 432 - 443
• Main Authors: Alsaker, Keith V, Paredes, Carlos J, Papoutsakis, Eleftherios T
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Nature B.V 01.10.2005
• Subjects: Batch culture; Data processing; Design; Design optimization; DNA chips; DNA microarrays; DNA probes; Gene expression; Gene regulation; Genes; Genetics; Genomes; Genomics; Homology; Hybridization; Polymerase chain reaction; Signal quality; Statistical analysis; Transcription; Transcriptomes
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/BF02989826

Microarray technology has contributed significantly to the understanding of bacterial genetics and transcriptional regulation. One neglected aspect of this technology has been optimization of microarray-generated signals and quality of generated information. Full genome microarrays were developed forClostridium acetobutylicum through spotting of PCR products that were designed with minimal homology with all other genes within the genome. Using statistical analyses it is demonstrated that signal quality is significantly improved by increasing the hybridization volume, possibly increasing the effective number of transcripts available to bind to a given spot, while changes in labeled probe amounts were found to be less sensitive to improving signal quality. In addition to Q-RT-PCR, array validation was tested by examining the transcriptional program of a mutant (M5) strain lacking the pSOL1 178-gene megaplasmid relative to the wildtype (WT) strain. Under optimal conditions, it is demonstrated that the fraction of false positive genes is 1% when considering differentially expressed genes and 7% when considering all genes with signal above background. To enhance genomic-scale understanding of organismal physiology, using data from these microarrays we estimated that 40-55% of theC. acetobutylicum genome is expressed at any time during batch culture, similar to estimates made forBacillus subtilis.[PUBLICATION ABSTRACT]