|
| Status |
Public on Feb 02, 2014 |
| Title |
Gene Expression Profile of Moraxella catarrhalis Grown in Pooled Human Sputum (Rep_2A) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Moraxella catarrhalis, grown in 20% pooled human sputum
|
| Organism |
Moraxella catarrhalis ATCC 43617 |
| Characteristics |
growth protocol: grown in 20% pooled human sputum in chemically-defined medium.
|
| Biomaterial provider |
ATCC
|
| Treatment protocol |
Bacterial cells were exposed to 20% pooled human sputum in chemically-defined medium.
|
| Growth protocol |
Bacterial cells were grown in 20% pooled human sputum in chemically-defined medium. The cells were grown at 37 degrees Celsius from and OD600 of 0.05 to a final OD600 of 0.9 and then harvested by centrifugation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The entire bacterial culture underwent total RNA extraction by utilization of the Ambion Ribopure Bacteria kit as per manufacturer's instructions and then were treated with DNase I from a MessageClean kit as per manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
Five micrograms of total RNA from the bacterial cells exposed to 20% pooled human sputum 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.
|
| |
|
| Channel 2 |
| Source name |
Moraxella catarrhalis, grown in chemically defined medium
|
| Organism |
Moraxella catarrhalis ATCC 43617 |
| Characteristics |
growth protocol: grown in chemically-defined medium.
|
| Biomaterial provider |
ATCC
|
| Treatment protocol |
Bacterial cells were exposed to chemically-defined medium.
|
| Growth protocol |
Bacterial cells were grown in chemically-defined medium. The cells were grown at 37 degrees Celsius from and OD600 of 0.05 to a final OD600 of 0.9 and then harvested by centrifugation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The entire bacterial culture underwent total RNA extraction by utilization of the Ambion Ribopure Bacteria kit as per manufacturer's instructions and then were treated with DNase I from a MessageClean kit as per manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Five micrograms of total RNA from the bacterial cells exposed to chemically defined medium 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.
|
| |
|
| |
| Hybridization protocol |
Equivalent amounts of the Cy5- and Cy3-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 Cy5- and Cy3-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 bacterial cells grown in chemically defined medium was utilized as the baseline gene expression template. To identify those genes that exhibited an altered expression profile upon exposure to pooled human sputum, a threshold of twofold-increased or decreased expression over the baseline was utilized.
|
| |
|
| Submission date |
Feb 03, 2011 |
| Last update date |
Feb 02, 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) |
| GSE27059 |
Gene Expression Profile of Moraxella catarrhalis Grown in Pooled Human Sputum |
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