Comment[ArrayExpressAccession] E-GEOD-43024 MAGE-TAB Version 1.1 Public Release Date 2013-10-10 Investigation Title Transcriptional profiles of Campylobacter jejuni NCTC 11168 to 0.125mg/L erythromycin treatment Comment[Submitted Name] Transcriptional profiles of Campylobacter jejuni NCTC 11168 to 0.125mg/L erythromycin treatment Experiment Description Erythromycin is the drug of choice to treat campylobacteriosis, but resistance to this antibiotic is rising. The adaptive mechanisms employed by Campylobacter jejuni to erythromycin treatment remain unknown. The aim of this study is to determine the molecular basis underlying CampylobacterM-bM-^@M-^Ys immediate response to Ery treatment. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni wild type strain NCTC 11168. One hybridizations were performed: sham-treated NCTC 11168 v.s. sub-lethal dose erythromycin treated NCTC 11168. Samples were independently grown and harvested. There were three biological replicates of each sample. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Xia Xia Wu Zhang Person First Name Qingqing Qingqing Zuowei Qijing Person Email carole_xia@yahoo.com.cn Person Affiliation Iowa State University Person Address Iowa State University, 1116 VMPM Iowa State University, Ames, IA, USA Person Roles submitter Protocol Name P-GSE43024-1 P-GSE43024-5 P-GSE43024-7 P-GSE43024-2 P-GSE43024-6 P-GSE43024-3 P-GSE43024-4 P-GSE43024-8 Protocol Description ImaGene software (BioDiscovery, El Segundo, CA) was used for spot finding and to measure the spot-specific mean signal strength of each spot. The fluorescentc intensities of all spots were log2 transformed. The median of the log2-transformed spot signals was determined and this value was used as spot signal for all further calculations. As an additional measure against intensity dependent dye bias the data was then subjected to LOWESS normalization. ID_REF = VALUE = normalized log2 ratio representing Ery treated/sham treated PRE_VALUE = normalized ratio representing Ery treated/sham treated cDNA was synthesized using aminoallyl-dNTP according to the manufacturerM-bM-^@M-^Ys instruction. After purification, aminoallyl labeled cDNA was fluorescently labeled with Cy-3/cy-5 mono-Reactive Dye Pack (Amersham Biosciences, Piscataway, NJ) After purification, the fluorescent labeling of the DNA was quantitated, and equal amounts of labeled cDNA of the wild-type and the mutant were mixed and dried in a speed vacuum concentrator. After resuspension with hybridization buffer provided by the slide manufacturer, the mixture was denatured by heating at 95 M-bM-^WM-&C for 10 min. The hybridization mixture was loaded onto a microarray slide and hybridized in a shaking incubator at 42 M-:C for 16 h. Slides were washed as the manufactureM-bM-^@M-^Ys recommendations and dried by centrifugation. Fifteen mL aliquots of NTCT 11168 culture (in duplicates) were treated with either sham (ethanol solvent for Ery), a sub-lethal dose of Ery (0.125 mg/L; 0.5x MIC). All cultures were thoroughly mixed and statically incubated under microaerobic conditions for 30 minutes at 42M-bM-^DM-^C. After 30 minutes treatment, the cultures were immediately mixed with RNAprotectM-bM-^DM-" (Qiagen, Valencia, CA) to stabilize the total bacterial RNA. Campylobacter strains were routinely cultured from frozen stocks (-80 M-bM-^DM-^C) on Mueller-Hinton (MH) agar or broth at 42M-bM-^DM-^C under microaerobic conditions (85% N2, 10% CO2 and 5% O2). wild-type strain NCTC 11168 was grown for 5 hours in MH to OD600 of approximately 0.2 with shaking (160 rpm) at 42oC under microaerobic condition. wild-type C. jejuni NCTC 11168 (Ery MIC: 0.25 mg/L) was grown for 5 hours in MH to OD600 of approximately 0.2 with shaking (160 rpm) at 42M-bM-^DM-^C under microaerobic condition. Total RNA was isolated using RNAprotectTM bacteria reagent (Qiagen) and an RNeasy kit (Qiagen) according to the manufacturerM-bM-^@M-^Ys protocol with an optional step of on-column DNase treatment with RNase-Free DNase (Qiagen). RNA concentration was measured with a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE) Using a ScanArray Express laser scanner (Applied Biosystem Inc., Foster City, CA) each dye channel of each slides was scanned at a 10 M-NM-