|
| Status |
Public on Jan 01, 2016 |
| Title |
AHLs vs. control rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
AHLs
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: CO92
variant: Pgm-
|
| Treatment protocol |
The overnight wild type cultures were washed twice with PBS buffer to remove the endogenous quorum sensing signals, the cells were diluted 1:100 in fresh culture medium, and then the cells were incubated for 2 hours at 37°C. Purified signals were then added to the cultures at the following concentrations: AHLs (5 μM N-(3-Oxooctanoyl)-L-homoserine lactone and N-Hexanoyl-DL-homoserine lactone). The control consisted of cells grown and treated under the same conditions without added signals. After 4 hours of induction, all of the cultures were centrifuged.
|
| Growth protocol |
An overnight culture was diluted 1:100 and grown at 37°C in BHI broth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were pelleted and immediately resuspended in 0.5 ml of RNA Protect Reagent (Qiagen, Valencia, CA). RNA was extracted from frozen cell pellets using the RNeasy Mini kit (Qiagen). The RNA was treated with DNase I (Ambion, Austin, TX) at 37°C for 30 min to remove the genomic DNA. The RNA was purified and concentrated by Microcon Ultracel/YM 30(Millipore, Billerica, MA).
|
| Label |
Cy3
|
| Label protocol |
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]).
|
| |
|
| Channel 2 |
| Source name |
control
|
| Organism |
Yersinia pestis |
| Characteristics |
strain: CO92
variant: Pgm-
|
| Treatment protocol |
The overnight wild type cultures were washed twice with PBS buffer to remove the endogenous quorum sensing signals, the cells were diluted 1:100 in fresh culture medium, and then the cells were incubated for 2 hours at 37°C. Purified signals were then added to the cultures at the following concentrations: AHLs (5 μM N-(3-Oxooctanoyl)-L-homoserine lactone and N-Hexanoyl-DL-homoserine lactone). The control consisted of cells grown and treated under the same conditions without added signals. After 4 hours of induction, all of the cultures were centrifuged.
|
| Growth protocol |
An overnight culture was diluted 1:100 and grown at 37°C in BHI broth.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were pelleted and immediately resuspended in 0.5 ml of RNA Protect Reagent (Qiagen, Valencia, CA). RNA was extracted from frozen cell pellets using the RNeasy Mini kit (Qiagen). The RNA was treated with DNase I (Ambion, Austin, TX) at 37°C for 30 min to remove the genomic DNA. The RNA was purified and concentrated by Microcon Ultracel/YM 30(Millipore, Billerica, MA).
|
| Label |
Cy5
|
| Label protocol |
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]).
|
| |
|
| |
| Hybridization protocol |
Microarray hybridization and post washes were performed using a Lucidea Slidepro Hybridization Station (GE Healthcare). Corresponding equal amounts (1.5 µg) 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 μl Long Oligo hybridization solution (Corning, Inc., Corning, NY), incubated at 95°C for 5 min, centrifuged (10,000 x g, 4 min), and kept at room temperature until injected into the hybridization station. Hybridization lasted for 16 hours at 42°C and washed in a series of wash buffers (2x saline-sodium citrate (SSC), 0.1% SDS; 1x SSC, and 0.1x SSC) by the hybridization station and dried by centrifugation at 1500 x g for 30 secs.
|
| Scan protocol |
Arrays scanned on a ProScanArray HT scanner (Perkin Elmer, Wellesley, MA) three times with varying photomultiplier tube gain and laser power settings.
|
| Description |
The strain Pgm- is pigmentation-negative mutant. The AHLs is the two AHL signals added in treatment.The control is the cells grown and treated under the same conditions without added three signals.
|
| Data processing |
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 0·05 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.
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| |
|
| Submission date |
Jul 09, 2010 |
| Last update date |
Jan 01, 2016 |
| Contact name |
Chris Minion |
| E-mail(s) |
fcminion@iastate.edu
|
| Phone |
515-294-6347
|
| Fax |
515-294-8500
|
| Organization name |
Iowa State University
|
| Department |
VMPM
|
| Street address |
1130 Vet Med
|
| City |
Ames |
| State/province |
IA |
| ZIP/Postal code |
50011 |
| Country |
USA |
| |
|
| Platform ID |
GPL10439 |
| Series (3) |
| GSE22847 |
Transcriptional analysis of AHLs signals add in experiment inYersinia pestis CO92 at 37°C |
| GSE22850 |
Outlier detection of biologically significant genes from combinatorial microarray data |
| GSE30373 |
Global gene expression analysis of *Yersinia pestis* AHL quorum sensing |
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