Genomic DNA standards for gene expression profiling in Mycobacterium tuberculosis.
A fundamental problem in DNA microarray analysis is the lack of a common standard to compare the expression levels of different samples. Several normalization protocols have been proposed to overcome variables inherent in this technology. As yet, there are no satisfactory methods to exchange gene expression data among different research groups or to compare gene expression values under different stimulus-response profiles. We have tested a normalization procedure based on comparing gene expression levels to the signals generated from hybridizing genomic DNA (genomic normalization). This procedure was applied to DNA microarrays of Mycobacterium tuberculosis using RNA extracted from cultures growing to the logarithmic and stationary phases. The applied normalization procedure generated reproducible measurements of expression level for 98% of the putative mycobacterial ORFs, among which 5.2% were significantly changed comparing the logarithmic to stationary growth phase. Additionally, analysis of expression levels of a subset of genes by real time PCR technology revealed an agreement in expression of 90% of the examined genes when genomic DNA normalization was applied instead of 29-68% agreement when RNA normalization was used to measure the expression levels in the same set of RNA samples. Further examination of microarray expression levels displayed clusters of genes differentially expressed between the logarithmic, early stationary and late stationary growth phases. We conclude that genomic DNA standards offer advantages over conventional RNA normalization procedures and can be adapted for the investigation of microbial genomes.
Talaat
Adel M
AM
Center for Biomedical Inventions and Department of Medicine, University of Texas-Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8573, USA.
Howard
Susan T
ST
Hale
Walker
W
Lyons
Rick
R
Garner
Harold
H
Johnston
Stephen Albert
SA
D004269:DNA, Bacterial / Q000032:analysis*; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D016680:Genome, Bacterial; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods / Q000592:standards*; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results
D004269:DNA, Bacterial; D012329:RNA, Bacterial