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Sample GSM348220 Query DataSets for GSM348220
Status Public on Dec 05, 2009
Title Hducreyi_FCS_Rep1
Sample type RNA
 
Channel 1
Source name H. ducreyi 35000HP, -FCS
Organism [Haemophilus] ducreyi 35000HP
Characteristics H. ducreyi 35000HP grown in Columbia broth for 8 hours.
Biomaterial provider Maria Labandeira-Rey
Treatment protocol Wild-type H. ducreyi strains were resuscitated from frozen stock on chocolate agar (CA) plates, and incubated at 33°C in a humidified atmosphere containing 95% air and 5% CO2 . Strains were grown at 33°C in a gyratory water bath at 100 rpm in a Columbia broth-based medium (35 g of Columbia broth (Difco) per liter, 0.1% (wt/vol) Trizma base (Sigma), equine hemin (25 µg/ml; Sigma), and 1% (vol/vol) IsoVitaleX (Becton Dickinson)).
Extracted molecule total RNA
Extraction protocol Stop buffer (200 mM Tris.Cl pH8, 20nM EDTA pH 8 and 20mM sodium azide) was added to 5-10 ml bacteria prior to collection by centrifugation. Total RNA was extracted using the RiboPure™ Kit (Ambion) following the manufacturer's instructions. After isolation, RNA was treated twice with DNaseI (Ambion) for 1 hr at 37°C, and purified using the RNeasy system (Qiagen). Samples were checked for residual DNA by PCR and re-treated with DNAseI if necessary.
Label Cy5
Label protocol Twenty micrograms of total RNA from cells grown in CB+FCS for 8 hrs, were used to generate cDNA with the Amino Allyl cDNA Labeling Kit (Ambion). After reverse transcription, residual RNA was removed by alkaline treatment, followed by neutralization. The cDNAs were purified using the QIAquick gel extraction kit (QIAGEN). After purification, Cy5 dye (GE Healthcare) was added following manufacturer's instructions. The labeled cDNAs were cleaned by using YM-30 Microcon centrifugal filter devices (Millipore) and labeling efficiency and concentration was determined using a NanoDrop spectophotometer.
 
Channel 2
Source name H. ducreyi 35000HP, +FCS
Organism [Haemophilus] ducreyi 35000HP
Characteristics H. ducreyi 35000HP grown in Columbia broth supplemented with 2.5% FCS, for 8 hours.
Biomaterial provider Maria Labandeira-Rey
Treatment protocol Wild-type H. ducreyi strains were resuscitated from frozen stock on chocolate agar (CA) plates, and incubated at 33°C in a humidified atmosphere containing 95% air and 5% CO2 . Strains were grown at 33°C in a gyratory water bath at 100 rpm in a Columbia broth-based medium (35 g of Columbia broth (Difco) per liter, 0.1% (wt/vol) Trizma base (Sigma), equine hemin (25 µg/ml; Sigma), 1% (vol/vol) IsoVitaleX (Becton Dickinson)) with 2.5% (vol/vol) heat-inactivated fetal calf serum (FCS).
Extracted molecule total RNA
Extraction protocol Stop buffer (200 mM Tris.Cl pH8, 20nM EDTA pH 8 and 20mM sodium azide) was added to 5-10 ml bacteria prior to collection by centrifugation. Total RNA was extracted using the RiboPure™ Kit (Ambion) following the manufacturer's instructions. After isolation, RNA was treated twice with DNaseI (Ambion) for 1 hr at 37°C, and purified using the RNeasy system (Qiagen). Samples were checked for residual DNA by PCR and re-treated with DNAseI if necessary.
Label Cy3
Label protocol Twenty micrograms of total RNA from cells grown in CB-FCS for 8 hrs, were used to generate cDNA with the Amino Allyl cDNA Labeling Kit (Ambion). After reverse transcription, residual RNA was removed by alkaline treatment, followed by neutralization. The cDNAs were purified using the QIAquick gel extraction kit (QIAGEN). After purification, Cy3 dye (GE Healthcare) was added following manufacturer's instructions. The labeled cDNAs were cleaned by using YM-30 Microcon centrifugal filter devices (Millipore) and labeling efficiency and concentration was determined using a NanoDrop spectophotometer.
 
 
Hybridization protocol Equal amounts of labeled cDNA from cells grown under both conditions were thoroughly mixed and used to hybridize microarray slides in the hybridization buffer provided with the CyScribe Post-Labeling kit (GE Healthcare). Hybridization was carried out at 50°C for 16 h in the dark.
Scan protocol After hybridization, the slides were washed in saline-sodium phosphate-EDTA buffer and scanned with GenePix scanner 4100A and GenePix Pro 5.0 software (Axon Instruments Inc.).
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-weighted scatterplot smoothing (LOWESS) normalization, which corrects intensity-dependent variation in dye bias, 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 in the cells grown in CB+FCS relative to cells grown in CB-FCS. The data were further scrutinized so as to only include expression profiles that were observed in at least three of the four experiments and had a P≤0.05 after one sample t-test analysis.
 
Submission date Dec 05, 2008
Last update date Dec 09, 2008
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)
GSE13851 H. ducreyi 35000HP grown in the presence or absence of FCS

Data table header descriptions
ID_REF
VALUE Log2 ratio of the medians (+FCS/-FCS)
F532 Median -FCS Signal median
F635 Median +FCS Signal median
B532 Median -FCS Background median
B635 Median +FCS Background median

Data table
ID_REF VALUE F532 Median F635 Median B532 Median B635 Median
1:01:01 -0.010134377 586.161 396.291 92.205 53.75
1:02:01 0.742437445 869.362 373.516 98.791 56.483
1:03:01 0.929033479 240.392 105.678 111.963 59.216
1:04:01 0.113700499 562.012 349.829 114.159 64.682
1:05:01 0.357270476 364.429 194.957 130.624 69.237
1:06:01 1.22897257 243.685 100.212 138.308 69.237
1:07:01 0.854394678 341.378 145.762 138.308 68.326
1:08:01 1.614945367 437.974 135.741 138.308 68.326
1:09:01 0.572404647 1656.398 769.807 142.698 69.237
1:10:01 0.429749851 2892.384 1468.555 151.48 68.326
1:11:01 1.25821739 2081.199 624.956 159.163 71.97
1:12:01 1.245495663 6897.815 2027.006 160.261 71.97
1:13:01 0.18142064 6713.405 4053.101 159.163 71.97
1:14:01 -0.365871442 3617.95 3142.998 163.554 76.525
1:15:01 -1.161653263 3953.84 5893.35 183.312 88.368
1:16:01 -1.282789701 3403.903 5471.55 199.778 102.945
1:01:02 -1.351074441 1002.181 1663.512 86.717 53.75
1:02:02 -1.095419565 1639.932 2332.196 94.4 55.572
1:03:02 -1.442222329 582.868 961.12 100.986 59.216
1:04:02 -0.805912948 220.633 195.868 108.67 61.038

Total number of rows: 5760

Table truncated, full table size 287 Kbytes.




Supplementary file Size Download File type/resource
GSM348220.gpr.gz 483.0 Kb (ftp)(http) GPR
Processed data included within Sample table

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