Genomic DNA standards for gene expression profiling in Mycobacterium tuberculosis

Adel M. Talaat 0 2 Susan T. Howard 0 1 2 Walker Hale IV 0 2 Rick Lyons 0 1 2 Harold Garner 0 2 Stephen Albert Johnston 0 2 0 Present address: Adel M. Talaat, Department of Animal Health and Biomedical Sciences, University of Wisconsin , Madison, WI 53706, USA 1 Department of Internal Medicine, University of New Mexico Health Science Center , 915 Camino de Salud, Albuquerque, NM 87131, USA 2 Center for Biomedical Inventions and Departments of Medicine, Microbiology and Biochemistry, University of Texas-Southwestern Medical Center , 5323 Harry Hines Boulevard, Dallas, TX 75390-8573, USA 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. - DNA microarray technology (oligo and spotted microarrays) has become widely accepted for gene expression profiling (1,2). There is a growing interest in applying such technologies to investigate the transcription profiles of infectious agents on a genome-wide level to develop new vaccines and drugs to combat infectious disease. A leading candidate for this approach is Mycobacterium tuberculosis, the causative agent of human tuberculosis, responsible for 3 million annual mortalities (3). However, microarray analysis has a number of problems, including spotting efficiency, sample labeling efficiency, transcript representation and hybridization reproducibility (4), which are amplified with the analysis of mixed RNA samples from infected tissues. We propose an alternative procedure for array hybridization that may circumvent some problems resulting from variables associated with DNA microarrays. Microarrays consist of in situ/pre-synthesized oligonucleotides or spotted cDNA representing all or a portion of expressed genes in an organism arrayed onto chemically treated glass slides or any other solid surface (5). Typically, transcripts from a variety of states are labeled with one of two dyes and pair-wise comparisons of relative changes in gene expression are estimated after co-hybridization to the same set of spotted arrays. There are a number of methods used to normalize these pair-wise comparisons. Current protocols for microarray data normalization use a control RNA sample from a particular tissue or time point (RNA normalization), a pool of grouped RNA samples from different tissues or different time points (6,7), or a subset of control reference genes (8) of known transcription profile. There are several problems with these approaches. For example, only genes with hybridization signals from both RNA samples can be used to generate relative expression levels. Signals observed from only one RNA sample are discarded. Under some growth conditions, the transcription levels of some genes will be undetectable (or very low), resulting in unmeasurable relative expression levels. Furthermore, for microbial systems, the grouped RNA normalization procedure may require pooling RNA from 20 or 30 experimental conditions at different growth phases. Comparisons of results to any new experimental condition would require a new control pool or a new set of hybridizations. Alternatively, using control genes for microarray data normalization is subject to the problem of choosing the right control genes, especially when even housekeeping genes can fluctuate under some experimental conditions (9). Even when a set of reference genes or an RNA pool is agreed upon for array analysis, the production of such control samples may vary from one experiment to another and from one laboratory to another. In response to these problems, we have explored an alternative procedure for array hybridization. In this procedure, hybridization signals from cDNA (prepared from total RNA) are normalized to signals generated from genomic DNA (gDNA) from the same organism. The proposed normalization protocol was applied to cultures of M.tuberculosis grown to either logarithmic or stationary phase. We found that a higher reproducibility and wider dynamic range are achievable using genomic normalization compared to an RNA normalization protocol. MATERIALS AND METHODS Construction of DNA microarrays We designed DNA microarrays by arraying an oligonucleotide set purchased from Operon Technologies (Alameda, CA), representing the whole genome of M.tuberculosis. The oligonucleotides were chosen using a proprietary algorithm (Operon Technologies) for selecting unique 70mers for each open reading frame (ORF) predicted in the published sequence of M.tuberculosis strain H37Rv (10) with an optimized melting temperature of 79 C (65 C). All oligonucleotides were resuspended in 33 SSC at a concentration of 40 mM using the liquid handling station Biomek 2000 (Beckman Coulter, Fullerton, CA). Resuspended oligonucleotides were spotted onto poly-L-lysine-coated glass slides (11) using a custom-built robotic arrayer (Magna Arrayer) assembled at the University of Texas Southwestern Medical Center (http://microarray.swmed.edu/technology.htm) that generates microarrays with a DNA spot size of 150200 mm in diameter. Sample preparation and slide hybridizations Mycobacterium tuberculosis H37Rv (ATCC no. 25618) was obtained from American Type Cell Culture. Logarithmic and stationary phase cultures for preparing RNA samples were grown in 7H9 medium supplemented with 10% OADC, at 37 C, and harvested at 14, 28 and 50 days. Total RNA was extracted from mycobacterial cultures in TRI reagent (Molecular Research Center, Cincinnati, O (...truncated)