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Sample GSM542692 Query DataSets for GSM542692
Status Public on May 03, 2011
Title Haemophilus ducreyi 35000HPcpxA relative to 35000HP sample 5
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 35000HPcpxA Signal median
B532 Median 35000HPcpxA Background median
F635 Median 35000HP Signal median
B635 Median 35000HP Background median

Data table
ID_REF VALUE F532 Median B532 Median F635 Median B635 Median
1:01:01 0.927517246 136.828 56.818 67.267 43.982
1:02:01 -0.267079618 189.008 56.818 133.671 45.707
1:03:01 -0.675765438 75.371 56.818 61.23 44.845
1:04:01 0.496717988 206.402 56.818 105.212 46.569
1:05:01 -0.841662973 105.52 60.297 93.139 48.294
1:06:01 -0.913216234 68.414 62.616 55.193 49.157
1:07:01 0.427606173 150.743 62.616 87.102 50.881
1:08:01 -1.03209363 229.593 64.935 238.021 51.744
1:09:01 -0.152003093 260.901 67.255 171.617 52.606
1:10:01 -0.666576266 285.252 67.255 244.92 53.468
1:11:01 -0.564904848 231.912 70.733 188.865 56.918
1:12:01 -0.678071905 1028.531 71.893 903.79 56.918
1:13:01 0.364012054 1600.194 69.574 713.201 55.193
1:14:01 0.838346737 1285.953 67.255 429.473 52.606
1:15:01 1.108357178 1301.027 61.457 366.518 48.294
1:16:01 0.953823954 1683.683 62.616 513.987 50.881
1:01:02 1.116364757 873.149 61.457 255.269 48.294
1:02:02 1.155101558 1074.913 60.297 300.113 48.294
1:03:02 1.631802755 350.187 60.297 100.038 48.294
1:04:02 1.378511623 124.073 59.138 60.368 46.569

Total number of rows: 5760

Table truncated, full table size 281 Kbytes.




Supplementary file Size Download File type/resource
GSM542692.gpr.gz 1.2 Mb (ftp)(http) GPR
Processed data included within Sample table

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