Comment[ArrayExpressAccession] E-GEOD-22848 MAGE-TAB Version 1.1 Public Release Date 2013-09-05 Investigation Title Transcription analysis of AI-2 signal add in experiment in Yersinia pestis CO92 at 37M-BM-0C Comment[Submitted Name] Transcription analysis of AI-2 signal add in experiment in Yersinia pestis CO92 at 37M-BM-0C Experiment Description Yersinia pestis, the etiological agent of plague, is able to sense cell density by quorum sensing. The function of quorum sensing in Y. pestis is not clear. Here, the process of autoinducer-2 (AI-2) quorum sensing was investigated by comparing transcript profiles when AI-2 quorum-sensing signal is added in. The strain M-bM-^HM-^Fpgm(pigmentation-negative) mutant R88 was used as wild type. The control consisted of cells grown and treated under the same conditions without added signals. Six independent RNA samples from Y. pestis CO92 M-bM-^HM-^Fpgm cultures were paired with six independent RNA samples from AI-2 signal added cultures for hybridization to six two-color microarrays. A dye-swap design was used to remove the Cy5 and Cy3 dye bias. 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 Minion Minion Yu Person First Name Chris Chris Jing Person Email fcminion@iastate.edu Person Affiliation Iowa State University Person Phone 515-294-6347 Person Fax 515-294-8500 Person Address VMPM, Iowa State University, 1130 Vet Med, Ames, IA, USA Person Roles submitter Protocol Name P-GSE22848-1 P-GSE22848-5 P-GSE22848-6 P-GSE22848-2 P-GSE22848-3 P-GSE22848-4 P-GSE22848-7 Protocol Description Background correction, compression, normalization and fitting each probe with a mixed model were conducted as previously described (Madsen ML, Nettleton D, Thacker EL, Minion FC. 2006. Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays. Microbiology 152:937-944 [PMID 16549658]) excluding slide region and slide-by-region interaction effects. Intensity = mu + trt + dye + slide Images were quantified using the softWorRx Tracker analysis software package (Applied Precision). Spot-specific mean signals were corrected for local background by subtracting spot-specific median background intensities. The natural logarithms of the background-corrected signals from a single scan were adjusted by an additive constant so that all scans of the same array-by-dye combination would have a common median. The median of these adjusted-log-background-corrected signals across multiple scans was then computed for each spot to obtain one value for each combination of spot, array and dye channel. These data for the two dye channels on any given array were normalized using locally weighted scatterplot smoother (LOWESS) normalization to adjust for intensity-dependent dye bias (Dudoit et al., 2000Down; Yang et al., 2002Down). Following LOWESS adjustment, the data from each channel were adjusted by an additive constant so that the median for any combination of array and dye would be the same for all array-by-dye combinations. The normalized values for triplicate spots were averaged within each array to produce one normalized measure of expression for each of the probe sequences and each of the RNA samples. A separate mixed linear model analysis was conducted for each probe sequence using the normalized data (Wolfinger et al., 2001Down). Each mixed model included fixed effects for treatment (iron depletion versus control), slide region (upper versus lower) and dye (Alexa 555 versus 647) as well as random effects for slide and slide-by-region interaction. A t-test for differential expression across treatments was conducted for each probe as part of our mixed linear model analyses. The P-values from these t-tests were converted to q-values using the method of Storey & Tibshirani (2003)Down. These q-values can be used to obtain approximate control of the false discovery rate at a specified value. For example, declaring probes with q-values less than or equal to 0M-BM-705 to be differentially expressed produces a list of significant results for which the false discovery rate is estimated to be approximately 5 %. Along with q-values, estimates of fold change were computed for each probe by taking the inverse natural log of the mean treatment difference estimated as part of our mixed linear model analyses. ID_REF = VALUE = Normalized log2 ratio: log(AI-2)-log(control) Target generation and labeling were performed as described (Carruthers MD and Minion C. 2009. Transcriptome analysis of Escherichia coli O157:H7 EDL933 during heat shock. FEMS Microbiol. Lett. 295:96-102 [PMID 19473256]). Microarray hybridization and post washes were performed using a Lucidea Slidepro Hybridization Station (GE Healthcare). Corresponding equal amounts (1.5 M-5g) of Cy3- or Cy5-labelled cDNA targets were mixed and dried using a Thermo Scientific Savant DNA SpeedVac Concentrator (Waltham, MA). Targets were suspended in 225 M-NM-