|
| Status |
Public on Jan 01, 2011 |
| Title |
Heat Shock 60 min replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C.burnetii Heat Shock 60 min
|
| Organism |
Coxiella burnetii |
| Characteristics |
strain: RSA 493 phase I
treatment: 35°C 9 days and 42°C 60 min
assayed molecule: bacteria mRNA
|
| Treatment protocol |
For temperature stress flasks containing L929 infected cells were incubated at 4°C and 42°C for 30 min and 1h with the Nine Mile I Strain.
|
| Growth protocol |
All experiments were performed with mid-log cultures of C. burnetii grown at 35°C on L929 cells with MEM medium (GIBCO, Invitrogen, Cergy-Pontoise, France) supplemented with 4% SVF (GIBCO) and 1% of L-Glutamine (GIBCO).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Infected cells were harvested using glass beads and centrifuged at 7.500 rpm for 10 min. Pellets were frozen using liquid nitrogen and stored at -80°C. Pellets were resuspended in 100 µl of TE supplemented with 10mg/ml of lyzozyme (Euromedex, Souffelweyersheim, France) and incubated for 10 min at room temperature. Total RNA was extracted and purified from resuspended pellets using the RNeasy mini Kit (Qiagen, Courtaboeuf, France) as recommended by the manufacturer. DNase treatment was performed using the DNA turbo free kit (Ambion, Applied Biosystems, Courtaboeuf, France). Total RNA integrity was checked using the 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA) and the concentrations were quantified using the NanoDrop (Thermo, Wingmilton, USA). Eukaryotic RNA was depleted using the MicrobEnrich kit as described (La MV 2007). The integrity of bacterial RNA was checked using the 2100 BioAnalyzer (Agilent) and the concentrations were quantified using the NanoDrop (Thermo Scientific).
|
| Label |
Cy3
|
| Label protocol |
RNA was retro-transcribed using M-MLV (Invitrogen, Cergy-Pontoise, France) and random hexamer primer (Invitrogen) as previously described (La MV 2007). cDNA were amplified by the processive polymerase phi29 using the GenomPhi illustrator V2 kit (GE HealthCare, Lifescience, Orsay, France) as described (La MV). The amplified cDNA were labeled with the Bioprime Labelling System (Invitrogen) using d-CTP Cy3/5 fluorochromes (GE HealthCare Lifescience). Labeled cDNA were purified using Qiaquick mini kit columns (Qiagen) and the level of incorporation was quantified using the NanoDrop (Thermo Scientific).
|
| |
|
| Channel 2 |
| Source name |
C.burnetii control 35°C
|
| Organism |
Coxiella burnetii |
| Characteristics |
strain: RSA 493 phase I
treatment: 35°C 9 days
assayed molecule: bacteria mRNA
|
| Treatment protocol |
For temperature stress flasks containing L929 infected cells were incubated at 4°C and 42°C for 30 min and 1h with the Nine Mile I Strain.
|
| Growth protocol |
All experiments were performed with mid-log cultures of C. burnetii grown at 35°C on L929 cells with MEM medium (GIBCO, Invitrogen, Cergy-Pontoise, France) supplemented with 4% SVF (GIBCO) and 1% of L-Glutamine (GIBCO).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Infected cells were harvested using glass beads and centrifuged at 7.500 rpm for 10 min. Pellets were frozen using liquid nitrogen and stored at -80°C. Pellets were resuspended in 100 µl of TE supplemented with 10mg/ml of lyzozyme (Euromedex, Souffelweyersheim, France) and incubated for 10 min at room temperature. Total RNA was extracted and purified from resuspended pellets using the RNeasy mini Kit (Qiagen, Courtaboeuf, France) as recommended by the manufacturer. DNase treatment was performed using the DNA turbo free kit (Ambion, Applied Biosystems, Courtaboeuf, France). Total RNA integrity was checked using the 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA) and the concentrations were quantified using the NanoDrop (Thermo, Wingmilton, USA). Eukaryotic RNA was depleted using the MicrobEnrich kit as described (La MV 2007). The integrity of bacterial RNA was checked using the 2100 BioAnalyzer (Agilent) and the concentrations were quantified using the NanoDrop (Thermo Scientific).
|
| Label |
Cy5
|
| Label protocol |
RNA was retro-transcribed using M-MLV (Invitrogen, Cergy-Pontoise, France) and random hexamer primer (Invitrogen) as previously described (La MV 2007). cDNA were amplified by the processive polymerase phi29 using the GenomPhi illustrator V2 kit (GE HealthCare, Lifescience, Orsay, France) as described (La MV). The amplified cDNA were labeled with the Bioprime Labelling System (Invitrogen) using d-CTP Cy3/5 fluorochromes (GE HealthCare Lifescience). Labeled cDNA were purified using Qiaquick mini kit columns (Qiagen) and the level of incorporation was quantified using the NanoDrop (Thermo Scientific).
|
| |
|
| |
| Hybridization protocol |
Hybridizations were carried out using two samples of labeled cDNA (75 pmol of each) that were labeled with Cy3 or Cy5 d-CTP. The pooled samples were hybridized using the GE hybridization kit (Agilent Technologies) as recommended by the manufacturer. The mixture was applied on a Surhyb 1 array (Agilent Technologies) and hybridized on the Coxiella burnetii array using the agilent hybridization chamber (Agilent Technologies). Microarrays were hybridized for 17h at 62°C in a rotative hoven. Microarrays were washed using the GE washing buffers (Agilent Technologies) with 5 min of Wash buffer 1 at room temperature followed by 1 min of Wash buffer 2 at 37°C. Microarrays were dried using a bath of acetonitrile (VWR, Fontenay sous Bois, France).
|
| Scan protocol |
The microarrays were scanned using the Agilent G2565C microarray scanner (Agilent Technologies) using XDR at 5µm resolution.
Images were quantified using Agilent Feature Extraction Software.
|
| Description |
Biological replicate 2 of 3; Control Vs Heat shock 60 min
|
| Data processing |
The data filtering and normalization were performed using Midas from the TM4 suite (TIGR). Data normalizations were performed using global normalization and lowess normalization methods. All experiments were conducted three times, which yielded 12 measurements per gene (representing four technical replicates in three biological replicates). Gene expression level was determined by determining the mean of the 12 values obtained for each probes.
The mean of Normalized data were processed using the Tmev software from the TM4 suite (TIGR) with a t-test with a p-value of <0.05 and a cut-off of 2 for the fold change
|
| |
|
| Submission date |
May 11, 2010 |
| Last update date |
Jan 01, 2011 |
| Contact name |
Kevin Lebrigand |
| Organization name |
IPMC/CNRS
|
| Lab |
Functional Genomics Platform of Nice-Sophia-Antipolis, France.
|
| Street address |
660 route des lucioles
|
| City |
Valbonne - Sophia-Antipolis |
| ZIP/Postal code |
06560 |
| Country |
France |
| |
|
| Platform ID |
GPL6675 |
| Series (1) |
| GSE21778 |
Early response of Coxiella burnetii to temperature stress |
|
