|
| Status |
Public on Mar 23, 2014 |
| Title |
Analysis of the Zur regulon of Moraxella catarrhalis rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Moraxella catarrhalis strain O35E, mid-log growth in BHI
|
| Organism |
Moraxella catarrhalis |
| Characteristics |
strain background: O35E
genotype/variation: wild type
|
| Biomaterial provider |
Eric J. Hansen
|
| Treatment protocol |
Bacterial cells were grown in BHI broth to mid-logarithmic phase.
|
| Growth protocol |
Bacterial cells were grown in BHI broth to mid-logarithmic phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by centrifugation and processed for total RNA with the Ambion RiboPure Bacteria kit.
|
| Label |
Cy5
|
| Label protocol |
Ten micrograms of total RNA from the O35E cells grown in BHI were mixed with 3.0 micrograms of M. catarrhalis genome directed primers and dried in the Speed-Vac. Once lyophilized the sample was reconstituted with 11 microliters of H2O and heated at 70 degrees Celsius for 5 minutes. The mixture was then cooled to room temperature for 10 minutes. cDNA synthesis was then performed with the Ambion Amino Allyl cDNA synthesis kit per the manufacturer's instructions RNA hydrolysis was accomplished with the addition of 15 microliters of sodium hydroxide (0.1M) and the sample was maintained at 70 degrees Celsius for 15 minutes. 15 microliters of hydrochloric acid (0.1M) was then added and the amino allyl-labeled cDNA was purified with a QIAGEN QIAquick gel extraction kit. After purification, Cy5 dye (GE Heathcare) was added and labelling occured per the manufacturers instructions. The CyDye-labeled cDNA sample was purified by using Microcon YM30 columns. A NanoDrop spectrophotometer was used to measure the concentration of the CyDye-labeled cDNA sample.
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| |
|
| Channel 2 |
| Source name |
Moraxella catarrhalis strain O35E non-polar ZUR mutant, mid-log growth in BHI
|
| Organism |
Moraxella catarrhalis |
| Characteristics |
strain background: O35E
genotype/variation: non-polar ZUR mutant
|
| Biomaterial provider |
Eric J. Hansen
|
| Treatment protocol |
Bacterial cells were grown in BHI broth to mid-logarithmic phase.
|
| Growth protocol |
Bacterial cells were grown in BHI broth to mid-logarithmic phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested by centrifugation and processed for total RNA with the Ambion RiboPure Bacteria kit.
|
| Label |
Cy3
|
| Label protocol |
Ten micrograms of total RNA from the O35E non-polar Zur mutant cells grown in BHI were mixed with 3.0 micrograms of M. catarrhalis genome directed primers and dried in the Speed-Vac. Once lyophilized the sample was reconstituted with 11 microliters of H2O and heated at 70 degrees Celsius for 5 minutes. The mixture was then cooled to room temperature for 10 minutes. cDNA synthesis was then performed with the Ambion Amino Allyl cDNA synthesis kit per the manufacturer's instructions RNA hydrolysis was accomplished with the addition of 15 microliters of sodium hydroxide (0.1M) and the sample was maintained at 70 degrees Celsius for 15 minutes. 15 microliters of hydrochloric acid (0.1M) was then added and the amino allyl-labeled cDNA was purified with a QIAGEN QIAquick gel extraction kit. After purification, Cy3 dye (GE Heathcare) was added and labelling occured per the manufacturers instructions. The CyDye-labeled cDNA sample was purified by using Microcon YM30 columns. A NanoDrop spectrophotometer was used to measure the concentration of the CyDye-labeled cDNA sample.
|
| |
|
| |
| Hybridization protocol |
Equivalent amounts of the Cy3- and Cy5-labeled cDNA samples were mixed, lyophilized, and then dissolved in 21 microliters of H2O. After denaturation for 3 minutes at 94 degrees Celsius these samples were kept on ice until they were used for DNA hybridization. The hybridization mixture containing 15 microliters of 4X hybridization buffer (GE Healthcare), 24 microliters of formamide (40% volume/volume), and 21 microliters of the Cy3- and Cy5-labeled cDNA. The mixture was added to a DNA microarray slide and incubated at 50 degrees Celsius overnight (~ 16 hours). After hybridization the slides were washed twice in 6X SSPE containing 0.01% Tween 20 at 50 degrees Celsius for 5 min, twice in 0.8X SSPE containing 0.001% Tween 20 at 50 degrees Celsius for 5 min, and twice in 0.8X SSPE at room temperature.
|
| Scan protocol |
The slides were dried by centrifugation at 900 x g in loosely capped 50-ml conical tubes for 3 minutes. Microarray slides were scanned with a GenePix 4100A microarray reader (Molecular Devices, Sunnyvale, CA) and analyzed with GenePix 4.0 software (Molecular Devices).
|
| Data processing |
The Acuity 4.0 software package (Molecular Devices) was utilized to analyze the microarray data. Raw data were first normalized using a ratio-based normalization method to equalize the means and medians of the features to 1 and exclude ratios that were less than 0.1 or greater than 10. Features that were flagged by the software as bad, absent, or not found were also excluded from further analysis. Secondly, a Lowess-based normalization was applied to the microarray data prior to final analysis. The RNA isolated from the in vitro broth-grown O35E wild-type cells was utilized as the baseline gene expression template. To identify those genes in the Zur mutant strain that exhibited an altered expression profile relative to the in vitro broth-grown wild-type cells , a threshold of twofold-increased or decreased expression over the baseline was utilized.
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| |
|
| Submission date |
Mar 23, 2011 |
| Last update date |
Mar 23, 2014 |
| Contact name |
Todd C. Hoopman |
| E-mail(s) |
[email protected]
|
| Phone |
214-648-5971
|
| Organization name |
University of Texas Southwestern Medical Center
|
| Department |
Microbiology
|
| Street address |
5323 Harry Hines Blvd.
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-9048 |
| Country |
USA |
| |
|
| Platform ID |
GPL7398 |
| Series (1) |
| GSE28151 |
Analysis of the Zur Regulon of Moraxella catarrhalis |
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