|
| Status |
Public on Oct 01, 2016 |
| Title |
Control unstressed VS Test acid-stressed bacterial cells_rep1.2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
US-C. jejuni bacterial cells
|
| Organism |
Campylobacter jejuni |
| Characteristics |
strain: 81-176
treatment: Unstressed
ph: pH 7 for 8 min
|
| Treatment protocol |
Total RNA was extracted using a hot phenol-chloroform method
|
| Growth protocol |
C. jejuni 81-176 was grown to logarithmic phase in biphasic MH medium (pH 7.4) under microaerophilic conditions. Bacterial cells were exposed to acid stress at pH 4 for 8 min in HCl-adjusted MH broth
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 μg of total RNA samples from each control (unstressed C. jejuni) and test sample (acid-stressed C. jejuni) were converted to cDNA using Superscript II (Invitrogen); 10 µg of random hexamers (Amersham Biosciences); a dNTP mixture of 0.5 mM dGTP, dATP and dCTP each; 0.16 mM dTTP; and 0.34 mM aminoallyl-dUTP. Aminoallyl-dUTP was incorporated in the reaction to permit the labeling of the cDNA with the monoreactive fluors indocarbocyanine (Cy3) and indodicarbocyanine (Cy5) (GE Healthcare)
|
| Label |
CY3
|
| Label protocol |
The aminoallyl-labeled cDNA was purified from free amines and unincorporated aminoallyl-dUTP by adding 350 μL of water and spinning through a Microcon YM-30 filter (Millipore) for 8 min at 10000 X g, followed by washing with sterile ddH2O. Following concentration and resuspension into NaHCO3 pH 9.0, the aminoallyl-labeled cDNA was coupled to either Cy3 (used to label control samples) or Cy5 dye (used to label test samples) by adding 10 μL of Cy3 or Cy5 (GE Healthcare) in dimethyl sulfoxide followed by incubation in the dark for 1 h at room temperature. Next, the fluorescently labeled cDNA was purified using QIAquick PCR purification kit according to the manufacturer’s instructions (Qiagen). Fluorescent Cy3- and Cy5-labeled cDNAs were combined and the fluor-labeled cDNA mix was dried under vacuum with a SpeedVac and resuspended in 15.14 μL of water, to which the following was added: 2.5 μL of salmon sperm DNA (10 mg/mL), 9 μL of 20X SSC, 0.36 μL of 10% SDS, and 9 μL of formamide.
|
| |
|
| Channel 2 |
| Source name |
AS-C. jejuni bacterial cells
|
| Organism |
Campylobacter jejuni |
| Characteristics |
strain: 81-176
treatment: Acid-stressed
ph: pH 4 for 8 min
|
| Treatment protocol |
Total RNA was extracted using a hot phenol-chloroform method
|
| Growth protocol |
C. jejuni 81-176 was grown to logarithmic phase in biphasic MH medium (pH 7.4) under microaerophilic conditions. Bacterial cells were exposed to acid stress at pH 4 for 8 min in HCl-adjusted MH broth
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10 μg of total RNA samples from each control (unstressed C. jejuni) and test sample (acid-stressed C. jejuni) were converted to cDNA using Superscript II (Invitrogen); 10 µg of random hexamers (Amersham Biosciences); a dNTP mixture of 0.5 mM dGTP, dATP and dCTP each; 0.16 mM dTTP; and 0.34 mM aminoallyl-dUTP. Aminoallyl-dUTP was incorporated in the reaction to permit the labeling of the cDNA with the monoreactive fluors indocarbocyanine (Cy3) and indodicarbocyanine (Cy5) (GE Healthcare)
|
| Label |
CY5
|
| Label protocol |
The aminoallyl-labeled cDNA was purified from free amines and unincorporated aminoallyl-dUTP by adding 350 μL of water and spinning through a Microcon YM-30 filter (Millipore) for 8 min at 10000 X g, followed by washing with sterile ddH2O. Following concentration and resuspension into NaHCO3 pH 9.0, the aminoallyl-labeled cDNA was coupled to either Cy3 (used to label control samples) or Cy5 dye (used to label test samples) by adding 10 μL of Cy3 or Cy5 (GE Healthcare) in dimethyl sulfoxide followed by incubation in the dark for 1 h at room temperature. Next, the fluorescently labeled cDNA was purified using QIAquick PCR purification kit according to the manufacturer’s instructions (Qiagen). Fluorescent Cy3- and Cy5-labeled cDNAs were combined and the fluor-labeled cDNA mix was dried under vacuum with a SpeedVac and resuspended in 15.14 μL of water, to which the following was added: 2.5 μL of salmon sperm DNA (10 mg/mL), 9 μL of 20X SSC, 0.36 μL of 10% SDS, and 9 μL of formamide.
|
| |
|
| |
| Hybridization protocol |
not provided
|
| Scan protocol |
The signal intensities of each spot were collected using ScanArray software (PerkinElmer).
|
| Description |
Unstressed contreol vs acid stressed test bacterial cells
|
| Data processing |
The spot intensities were normalized via locally weighted linear regression (LOWESS) using MIDAS
|
| |
|
| Submission date |
Oct 06, 2015 |
| Last update date |
Oct 01, 2016 |
| Contact name |
ALAIN STINTZI |
| E-mail(s) |
astintzi@uottawa.ca
|
| Phone |
6135625800
|
| Organization name |
UOTTAWA
|
| Department |
BIOCHEMSISTRY, MICROBIOLOGY AND IMMUNOLOGY
|
| Lab |
DR ALAIN STINTZI LAB
|
| Street address |
451 SMYTH ROAD
|
| City |
ottawa |
| State/province |
ONTARIO |
| ZIP/Postal code |
K1H 8M5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6293 |
| Series (1) |
| GSE73793 |
Exposure of Campylobacter jejuni to acid stress enhances its pathogenesis and cross-protects the bacterium against other stresses |
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