|
| Status |
Public on Jan 31, 2010 |
| Title |
Heat-killed M. bovis infected for two hours vs live M. bovis infected for two hours 3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Alveolar macrophages
|
| Organism |
Bos taurus |
| Characteristics |
animal number: 202429
cell type: Alveolar macrophages
infection type: Heat-killed M. bovis
infection time: 2 hours
|
| Treatment protocol |
Cells were uninfected or infected with either live or heat-killed M. bovis for two or four hours.
|
| Growth protocol |
Alveolar macrophages harvested from cattle aged 6-12 months, cells cultured in RPMI-1640 + Glutamax, 10% heat-inactivaled foetal calf serum (FCS), 5x 10(5) beta-mercaptoethanol, 1x penicillin/streptomycin, 2.5μg/ml fungizone for 24 hours, media then changed to as before except 3% FCS and no penicillin/streptomycin and cultured for a further 24 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells lysed with 4M guanidine thiocyantae, homogenised using 20g needle and bacteria removed by pelleting. RNA then extracted using Qiagen Rneasy Maxi kit according to manufacturer's instructions.
|
| Label |
Alexa Fluor 647
|
| Label protocol |
10μg total RNA reverse transcribed using Superscript Plus Indirect cDNA labelling system (Invitrogen) with Oligo dT primers. cDNA was labelled with Alexa Fluor dyes 555 and 647.
|
| |
|
| Channel 2 |
| Source name |
Alveolar macrophages
|
| Organism |
Bos taurus |
| Characteristics |
animal number: 202429
cell type: Alveolar macrophages
infection: Live M. bovis
infection time: 2 hours
|
| Treatment protocol |
Cells were uninfected or infected with either live or heat-killed M. bovis for two or four hours.
|
| Growth protocol |
Alveolar macrophages harvested from cattle aged 6-12 months, cells cultured in RPMI-1640 + Glutamax, 10% heat-inactivaled foetal calf serum (FCS), 5x 10(5) beta-mercaptoethanol, 1x penicillin/streptomycin, 2.5μg/ml fungizone for 24 hours, media then changed to as before except 3% FCS and no penicillin/streptomycin and cultured for a further 24 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells lysed with 4M guanidine thiocyantae, homogenised using 20g needle and bacteria removed by pelleting. RNA then extracted using Qiagen Rneasy Maxi kit according to manufacturer's instructions.
|
| Label |
Alexa Fluor 555
|
| Label protocol |
10μg total RNA reverse transcribed using Superscript Plus Indirect cDNA labelling system (Invitrogen) with Oligo dT primers. cDNA was labelled with Alexa Fluor dyes 555 and 647.
|
| |
|
| |
| Hybridization protocol |
cDNA was resuspended in SlideHyb™ Glass Array Hybridization buffer #3 (Ambion). Pre-processing, hybridization, for 3hrs at 65°C, 3 hrs at 55°C and 12hrs at 50°C, and washing was performed in a Tecan HS400™ Hybridization station.
|
| Scan protocol |
Scanned on a GenePix 4000A scanner.
|
| Description |
Biological replicate 4 of 5
|
| Data processing |
Data were processed using the R package limma. Background was subtracted and loess normalization applied.
|
| |
|
| Submission date |
Dec 01, 2009 |
| Last update date |
Dec 01, 2009 |
| Contact name |
Michael Bryan Watson |
| E-mail(s) |
mick.watson@roslin.ed.ac.uk
|
| Organization name |
The Roslin Institute
|
| Department |
ARK-Genomics
|
| Street address |
Roslin Biocentre
|
| City |
Roslin |
| State/province |
Midlothian, EH25 9PS |
| ZIP/Postal code |
EH25 9PS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL5751 |
| Series (1) |
| GSE19237 |
Early response of bovine alveolar macrophages to infection with live and heat-killed Mycobacterium bovis |
|
