|
| Status |
Public on Nov 04, 2013 |
| Title |
Haemophilus ducreyi 35000HPfis relative to 35000HP rep5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Haemophilus ducreyi 35000HP grown in Columbia broth supplemented with 2.5% FCS for 8 hr
|
| Organism |
[Haemophilus] ducreyi |
| Characteristics |
strain: 35000HP
|
| Treatment protocol |
After the fis mutant was generated, both strains were grown in Columbia Broth. After 8 hr of growth RNA was isolated and processed for DNA microarray analysis. This study includes three biological replicates (paired samples), and all three were subjected to dye swap.
|
| 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 |
Ten 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 35000HPfis grown in Columbia broth supplemented with 2.5% FCS for 8 hr
|
| Organism |
[Haemophilus] ducreyi |
| Characteristics |
strain: 35000HPfis
|
| Treatment protocol |
After the fis mutant was generated, both strains were grown in Columbia Broth. After 8 hr of growth RNA was isolated and processed for DNA microarray analysis. This study includes three biological replicates (paired samples), and all three were subjected to dye swap.
|
| 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 |
Ten 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
|
| |
|
| |
| Hybridization protocol |
Equal amounts of labeled cDNA from 35000HP or 35000HPfis 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 washed in saline-sodium phosphate EDTA buffer and scanned with GenePix 4100A scanner and analyzed with GenePix Pro 5.0 software (Axon instruments)
|
| 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 35000HPfis 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 |
Feb 19, 2013 |
| Last update date |
Nov 05, 2013 |
| 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) |
| GSE44413 |
Haemophilus ducreyi 35000HPfis relative to 35000HP |
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