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| Status |
Public on May 23, 2009 |
| Title |
Non-Infected Vero cells day 7 25C rep 5 |
| Sample type |
RNA |
| |
|
| Source name |
Non-Infected Vero cells day 7 25C rep 5
|
| Organism |
Rickettsia rickettsii |
| Characteristics |
genotype: R strain
|
| Treatment protocol |
To investigate the transcriptional response of R. rickettsii exposed to limiting iron conditions, monolayers of Vero cells in T25 tissue culture flasks were infected with R. rickettsii R strain at an MOI of 0.025. To mimic iron limitation R. rickettsii were grown for three days at 34°C at which time 200 μM of deferozamine mesylate was added to the flasks and incubated for 24 hours. Total RNA was extracted by removing the growth media and adding 1 ml of Trizol (Invitrogen) to thee T25 tissue culture flasks grown for three days and to three T25 tissue culture flasks exposed to iron limiting conditions for 24 hrs. To examine gene expression at different growth temperatures monolayers of Vero or ISE6 cells in T25 tissue culture flasks were infected with R. rickettsii R strain at an MOI of 0.025. Total RNA was extracted as above from five T25 tissue culture flasks grown at 37°C or 34°C for two and three days after infection and total RNA from five T25 tissue culture flasks grown at 25°C or 22°C for six and eight days following infection. To study cold shock response in R. rickettsii 15 T25 flasks were infected with an MOI of 0.025 and incubated at 34°C for three days. After three days of growth five flasks were removed for isolation of total RNA and the remainder were shifted to 4°C for 2 hrs. After 2 hrs, total RNA from 5 flasks was extracted and the remaining five flasks were shifted back to 34°C for 2 hrs before total RNA was extracted. In a separate experiment six T25 tissue culture flasks were infected with an MOI of 0.025 and grown for three days at 34°C. As above total RNA was extracted from three T25 tissue culture 18 flasks on day three after infection and three T25 tissue culture flasks were moved to 4°C for 24 hrs before extraction. For iron limitation, growth temperature and cold shock plaque forming units (PFUs) were determined to ensure equal numbers of rickettsiae were being compared in each experiment. To determine non-specific background from ISE6 and Vero RNA, RNA was harvested from uninfected samples and hybridized to the microarrays. These experiments showed a false positive rate of 2 to 5%. These genes were removed from the final analysis.
|
| Growth protocol |
R. rickettsii R strain was propagated in Vero cells with M199 medium or ISE6 tick cells (Munderloh et al., 1999) with L-15B medium and purified by Renografin density gradient centrifugation (Weiss et al., 1975). To determine the growth rate of R. rickettsii R strain grown at different temperatures, R. rickettsii was grown on monolayers of either ISE6 or Vero cells in T25 flasks (MOI of 0.025). For the growth rates of R. rickettsii R strain under limiting iron conditions, rickettsiae were grown on monolayers of Vero cells (MOI of 0.025) in 6 well plates with 0, 100, 200 and 500 μM concentrations of deferozamine mesylate (Sigma). To restore growth in the presence of 200 or 500 μM deferozamine mesylate, iron saturated holotransferrin (Sigma) was included at concentrations of 12 and 24 mg/ml respectively. At the appropriate times after infection, the medium was removed and 1 ml of K36 (0.1 M KCl, 0.015 M NaCl, 0.05 M K2HPO4, 0.05 M KH2PO4, pH 7.0) was added. The monolayer was removed by scraping and the host cells were lysed by bead beating 2X for 4 seconds with (1 mm) glass beads. The cell suspension was then frozen at -80°C until plaque assays were performed. Plaque assays were performed as 17 previously described (Cory et al., 1974). All growth curves are representative of multiple experiments and each point is an average of at least two replicate platings.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from each sample was stabilized with RNAProtect (Qiagen), extracted and purified using the 96-well plate RNAeasy kit (Qiagen) with slight modifications. RNA samples were treated with DNA-free DNase I (Ambion) and the quality of DNA-free RNAs were monitored on Agilent RNA Pico II LabChips (Agilent Technologies). Additionally, possible DNA contamination was tested by qPCR (see below). 10 mg of total RNA from each sample was reverse-transcribed to cDNA using random hexamer primers (Invitrogen) and SuperScript III reverse transcriptase (Invitrogen). RNA was removed and cDNA was purified using a QiaQuick 96 PCR purification kit (Qiagen), quantified and fragmented with DNase I (Roche)
|
| Label |
biotin
|
| Label protocol |
Enzo BioArray Terminal Labeling Kit with Biotin-ddUTP (Affymetrix) was used to label the 3′ termini of the fragmented cDNA products. Labeled cDNA (1 µg) was hybridized to custom Affymetrix GeneChips
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| |
|
| Hybridization protocol |
~1ug label and fragmented applied for 16hrs 40OC
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip 3000 7GPlus Scanner.
|
| Description |
Gene expression data from_Non-Infected Vero cells day 7 25C rep 5
|
| Data processing |
The data were analyzed with GCOS 1.4 using Affymetrix default analysis with a scale filter for Ft genes setting and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
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| |
|
| Submission date |
Feb 24, 2009 |
| Last update date |
May 23, 2009 |
| Contact name |
Dan Sturdevant |
| E-mail(s) |
dsturdevant@niaid.nih.gov
|
| Phone |
4063639248
|
| Organization name |
NIH
|
| Department |
NIAID
|
| Lab |
RTS
|
| Street address |
903 S 4th street
|
| City |
Hamilton |
| State/province |
MT |
| ZIP/Postal code |
59840 |
| Country |
USA |
| |
|
| Platform ID |
GPL4692 |
| Series (1) |
| GSE14965 |
Limited transcriptional responses of Rickettsia rickettsii exposed to environmental stimuli |
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