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Sample GSM542690 Query DataSets for GSM542690
Status Public on May 03, 2011
Title Haemophilus ducreyi 35000HPcpxA relative to 35000HP sample 3
Sample type RNA
 
Channel 1
Source name Haemophilus ducreyi 35000HP
Organism [Haemophilus] ducreyi
Characteristics strain: 35000HP
genotype/variation: wild type
Biomaterial provider Maria Labandeira-Rey
Treatment protocol Cells are grown under normal conditions
Growth protocol Haemophilus ducreyi strain was resucitated from frozen stock on Chocolate agar plates, and incubated ON at 33C in a humidified atmosphere containing 95% air and 5% CO2. Columbia broth supplemented with 2.5% FCS is inoculated from the plate and cultures are grown in a 33C gyratory water bath at 100 rpm for 8 hrs
Extracted molecule total RNA
Extraction protocol Total RNA from 2-4 ml of culture was extracted using the Ribopure Bacteria kit (Ambion) following manufacturer's instructions. After isolation, RNA was treated twice with DNaseI (NEB) for 1 hr at 37C, and purified using RNeasy system (Qiagen). Samples were checked for residual DNA by PCR and re-treated if necessary. Quality assessment was performed using the Agilent 2100 Bioanalyzer (Agilent Technologies)
Label Cy3
Label protocol Twenty micrograms of total RNA for cells grown in Columbia broth for 8 hr were used to generate cDNA using the Amino Allyl cDNA Labeling kit ragents (Ambion). After reverse transcription, residual RNA was removed by alkaline treatment, followed by neutralization. The cDNAs were purified using the Qiaquick gel extraction columns (Qiagen). After purification, Cy3 dye (GE Healthcare) was added following manufacturer's instructions. The labeled cDNAs were cleaned by using YM-30 Microcon centrifugal devices (Millipore) and labeling efficiency was determined using a Nanodrop spectrophotometer.
 
Channel 2
Source name Haemophilus ducreyi 35000HPcpxA
Organism [Haemophilus] ducreyi
Characteristics strain: 35000HP
genotype/variation: cpxA deletion mutant
Biomaterial provider Maria Labandeira-Rey
Treatment protocol Cells are grown under normal conditions
Growth protocol Haemophilus ducreyi strain was resucitated from frozen stock on Chocolate agar plates, and incubated ON at 33C in a humidified atmosphere containing 95% air and 5% CO2. Columbia broth supplemented with 2.5% FCS is inoculated from the plate and cultures are grown in a 33C gyratory water bath at 100 rpm for 8 hrs
Extracted molecule total RNA
Extraction protocol Total RNA from 2-4 ml of culture was extracted using the Ribopure Bacteria kit (Ambion) following manufacturer's instructions. After isolation, RNA was treated twice with DNaseI (NEB) for 1 hr at 37C, and purified using RNeasy system (Qiagen). Samples were checked for residual DNA by PCR and re-treated if necessary. Quality assessment was performed using the Agilent 2100 Bioanalyzer (Agilent Technologies)
Label Cy5
Label protocol Twenty micrograms of total RNA for cells grown in Columbia broth for 8 hr were used to generate cDNA using the Amino Allyl cDNA Labeling kit ragents (Ambion). After reverse transcription, residual RNA was removed by alkaline treatment, followed by neutralization. The cDNAs were purified using the Qiaquick gel extraction columns (Qiagen). After purification, Cy5 dye (GE Healthcare) was added following manufacturer's instructions. The labeled cDNAs were cleaned by using YM-30 Microcon centrifugal devices (Millipore) and labeling efficiency was determined using a Nanodrop spectrophotometer.
 
 
Hybridization protocol Equal amounts of labeled cDNA from 35000HP or 35000HPcpxA were thoroughly mixed and used to hybridize microarray slides in Amersham microarray hybridization buffer. Hybridization was carried out at 50C for 16 hr in the dark.
Scan protocol After hybridization, the slides were washe in saline-sodium phosphate EDTA buffer and scanned with GenePix 4100A scanner and analyzed with GenePix Pro 5.0 software (Axon instruments)
Description n/a
Data processing Data was subjected to two types of normalization, a ratio-based normalization so that the mean or median intensities are the same across the array, and a non-linear locally weighed scatterplot smoothing (LOWESS) normalization, which corrects intensity dependent variation in dye basis, before being combined into a single data set for further analysis. Differential expression was defined as a minimum of two fold over or under expression of 35000HPcpxA relative to 35000HP. The data were further scrutinized so as to only include expression profiles that were observed in at least 4 of the 6 experiments and had a p<0.05 after one t-test analysis.
 
Submission date May 11, 2010
Last update date May 03, 2011
Contact name Eric J Hansen
E-mail(s) eric.hansen@utsouthwestern.edu
Phone 2146331386
Organization name University of Texas Southwestern Medical Center
Department Microbiology
Lab Eric J. Hansen
Street address 5323 Harry Hines Blvd
City Dallas
State/province TX
ZIP/Postal code 75390-9048
Country USA
 
Platform ID GPL7741
Series (1)
GSE21788 Haemophilus ducreyi 35000HPcpxA gene expression relative to 35000HP

Data table header descriptions
ID_REF
VALUE Log2 ratio of the medians (35000HPcpxA/35000HP)
F532 Median 35000HP Signal median
B532 Median 35000HP Background median
F635 Median 35000HPcpxA Signal median
B635 Median 35000HPcpxA Background median

Data table
ID_REF VALUE F532 Median B532 Median F635 Median B635 Median
1:01:01 null 0 0 0 0
1:02:01 null 0 0 0 0
1:03:01 -0.562772261 35.251 34.036 23.857 23.035
1:04:01 1.021479727 40.113 34.036 36.197 23.857
1:05:01 0.552868871 42.544 35.251 33.729 23.035
1:06:01 0.021479727 37.682 35.251 26.325 23.857
1:07:01 0.437227739 38.898 35.251 29.616 24.68
1:08:01 -0.241270432 44.976 35.251 32.907 24.68
1:09:01 0.690640579 52.269 36.467 51.005 25.503
1:10:01 0.495695163 51.053 36.467 46.069 25.503
1:11:01 0.173767068 47.407 36.467 37.843 25.503
1:12:01 0.109694865 114.262 36.467 109.415 25.503
1:13:01 0.69777382 100.891 36.467 129.159 24.68
1:14:01 1.003602237 69.287 36.467 90.494 24.68
1:15:01 1.602171791 80.227 37.682 154.662 25.503
1:16:01 1.653977179 85.089 36.467 178.519 25.503
1:01:02 null 0 0 0 0
1:02:02 null 0 0 0 0
1:03:02 1.758729568 43.76 34.036 55.941 23.035
1:04:02 1.244278199 36.467 34.036 28.793 23.035

Total number of rows: 5760

Table truncated, full table size 265 Kbytes.




Supplementary file Size Download File type/resource
GSM542690.gpr.gz 730.0 Kb (ftp)(http) GPR
Processed data included within Sample table

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