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Sample GSM542698 Query DataSets for GSM542698
Status Public on May 03, 2011
Title Haemophilus ducreyi 35000HPcpxRpML125 relative to 35000HPcpxRpLS88 sample 1
Sample type RNA
 
Channel 1
Source name Haemophilus ducreyi 35000HPcpxRpLS88
Organism [Haemophilus] ducreyi
Characteristics strain: 35000HP
genotype/variation: cpxRpLS88 (vector control)
Biomaterial provider Maria Labandeira-Rey
Treatment protocol Strains were 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 35000HPcpxRpML125
Organism [Haemophilus] ducreyi
Characteristics strain: 35000HP
genotype/variation: cpxRpML125 (over-expresses CpxR in a CpxR deletion background)
Biomaterial provider Maria Labandeira-Rey
Treatment protocol Strains were 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)
GSE21789 Global gene expression profile of Haemophilus ducreyi 35000HPcpxRpML125 relative to 35000HPcpxRpLS88

Data table header descriptions
ID_REF
VALUE Log2 ratio of the medians 35000HPcpxRpML125 relative to 35000HPcpxRpLS88
F635 Median 35000HPcpxRpML125 Signal median
B635 Median 35000HPcpxRpML125 Background median
F532 Median 35000HPcpxRpLS88 Signal median
B532 Median 35000HPcpxRpLS88 Background median

Data table
ID_REF VALUE F635 Median B635 Median F532 Median B532 Median
1:01:01 1.02644598 89 34 57 30
1:02:01 0.751892138 67 35 50 31
1:03:01 0.41467678 39 35 34 31
1:04:01 -0.16326792 61 36 61 33
1:05:01 0.41467678 49 37 42 33
1:06:01 -1 39 37 38 34
1:07:01 0.09895848 53 38 49 35
1:08:01 0.447843644 53 38 46 35
1:09:01 -0.321928095 74 38 79 34
1:10:01 0.041242982 73 38 68 34
1:11:01 -0.06340917 60 38 57 34
1:12:01 -0.562772261 126 38 164 34
1:13:01 -0.341902795 173 38 205 34
1:14:01 -0.60823228 164 38 226 34
1:15:01 -0.395928676 170 37 208 33
1:16:01 -0.332789088 204 38 242 33
1:01:02 -0.06039728 198 35 200 30
1:02:02 -0.360304767 261 35 321 31
1:03:02 -0.351074441 104 35 119 31
1:04:02 -0.298672743 48 35 48 32

Total number of rows: 5760

Table truncated, full table size 179 Kbytes.




Supplementary file Size Download File type/resource
GSM542698.gpr.gz 790.0 Kb (ftp)(http) GPR
Processed data included within Sample table

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