Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Mhyo IG Rnd vs MhRnd

ABSTRACT: Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist. The virulence factors of M. hyopneumoniae are largely unknown and are most probably complex in nature. The transcriptional profile of intergenic regions is investigated using microarray PCR and oligonucleotide probes. It is hypothesized that intergenic regions of the Mhyo genome are being transcribed along with the ORFs. To test for transcripts in IG regions, cDNA was generated from M. hyopneumoniae RNA using random hexamers (IDT; Integrated DNA Technologies, Inc. Coralville, IA) and a set of 129 hexamer oligonucleotide primers designed for M. hyopneumoniae mRNA. A single total RNA sample was split into eight 5 μg samples, and cDNA synthesis was conducted on four of these samples using the random hexamers (IDT); for the other four samples, cDNA was generated using primers designed for M. hyopneumoniae as described by Madsen et al. 2006. In each set of four, two samples were fluorescently labeled with Cy3 and the other two were labeled with Cy5 using an indirect labeling protocol. After labeling and cleanup, all 8 samples were quantified on a Nanodrop ND-1000 spectrophotometer. For each array on the slide, 1 μg of labeled cDNA from each type of primer generation (Cy3 or Cy5, random or specific primer set) was combined and hybridized to a PCR featured array and an oligonucleotide featured array. This resulted in 2 arrays of PCR probes hybridized with dye swaps and 2 arrays of oligonucleotide probes with dye swaps. A second RNA sample was split in four 5 μg samples of total RNA, and the hybridization process was repeated for the oligonucleotide probe array.

ORGANISM(S): Mycoplasma hyopneumoniae

SUBMITTER: Chris Minion

PROVIDER: E-GEOD-17893 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays.

Madsen Melissa L ML Nettleton Dan D Thacker Eileen L EL Edwards Robert R Minion F Chris FC

Infection and immunity 20060101 1

Bacterial pathogens undergo stress during host colonization and disease processes. These stresses result in changes in gene expression to compensate for potentially lethal environments developed in the host during disease. Mycoplasma hyopneumoniae colonizes the swine epithelium and causes a pneumonia that predisposes the host to enhanced disease from other pathogens. How M. hyopneumoniae responds to changing environments in the respiratory tract during disease progression is not known. In fact, ...[more]

PMID: 16368969

Publication: 1/2

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Project description:Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist although it is clear that M. hyopneumoniae adheres to porcine ciliated epithelium by action of a protein called P97. Previous studies have shown variation in the gene encoding the P97cilium adhesin within different strains of M. hyopneumoniae, but the extent of genetic variation among field strains across the genome is not known. Since M. hyopneumoniae is a worldwide problem, it is reasonable to expect that a wide range of genetic variability may exist given all of the different breed and housing conditions. This variation may impact the overall virulence of a single strain. Using microarray technology, this study examined potential variation of fourteen field strains in comparison to strain 232 on which the array was based. Genomic DNA was obtained, amplified with TempliPhiâ¢, and labeled indirectly with Alexa dyes. Post genomic hybridization, the arrays were scanned and data analyzed using a linear statistical model. Results indicate that genetic variation could be detected in all fourteen field strains but across different loci, suggesting that variation occurs throughout the genome. Fifty-nine percent of the variable loci were hypothetical genes. Twenty-two percent of the lipoprotein genes showed variation in at least one field strain. A permutation test identified a location in M. hyopneumoniae genome where spatial clustering of variability between the field strains and strain 232 exists. TempliPhiâ¢ amplified DNA samples from field isolates were compared to control strain 232 using a two-color experimental microarray design. Independent samples from one isolate labeled with one dye were paired with control samples labeled with the alternate dye; the samples were mixed and hybridized to the microarray. For nine of the fourteen isolates (95MP1501, 95MP1502, 95MP1503, 95MP1504, 95MP1508, 95MP1509, 97MP0001, 00MP1301, and 05MP2301), four independent field isolate DNA samples were paired with four independent DNA samples from control 232. In two of the four arrays, the control sample was labeled with Alexa 555 dye and compared to the field isolate sample labeled with Alexa 647 dye (Molecular Probes, Inc., Eugene, Ore.). The dye assignment to control and treated samples was reversed for the other two arrays (dye swap). The arrays were hybridized under identical conditions as described below. This procedure was repeated for isolates 95MP1510 for a total of four arrays, where the control sample was labeled with Alexa 647 dye in three of the arrays and Alexa 555 dye for the fourth array. For isolates 95MP1505, 95MP1506 and 95MP1507, a total of five arrays each, including two dye swaps, were done; and for isolate 00MP1502, a total of six arrays were done with the control labeled with Alexa 555 dye for four of the arrays and Alexa 647 for the other two arrays.

Dataset Information

Mhyo IG Rnd vs MhRnd

Publications

Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays.

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