|
| Status |
Public on Dec 05, 2009 |
| Title |
Hducreyi_FCS_Rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| 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 |
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 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 |
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 |
|
