|
| Status |
Public on Sep 04, 2014 |
| Title |
CAM explants infected with wild type B. abortus 2308: Replicate 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
bovine trophoblastic cells [test]
|
| Organism |
Bos taurus |
| Characteristics |
source tissue: intact pregnant bovine uteruses at the final third of gestation
tissue: Chorioallantoic membrane explant culture (CAM)
cell type: trophoblastic cells
infection: wild type B. abortus 2308
infection time: 4 hours
|
| Treatment protocol |
The trophoblastic surface of the CAM explants was inoculated with 2.0 x 107 CFU, in a multiplicity of infection of approximately 1000 (MOI 1000:1). CAM explants were inoculated in triplicates with wild type, ΔvirB2, or ΔbtpB B. abortus 2308. Plates were incubated at 37°C with 5% CO2 for 4 h, the medium was then replaced with medium supplemented with 50 µg gentamicin/mL (Invitrogen, São Paulo, Brazil) to inactivate extracellular bacteria. Plates were maintained at 37°C with an atmosphere containing 5% CO2 for 1 h followed by washing three times with with PBS (phosphate buffered saline - pH 7.4) to eliminate the antibiotic. Uninfected CAM controls were inoculated with sterile medium and kept under the same conditions
|
| Growth protocol |
Seven intact pregnant bovine uteruses at the final third of gestation were obtained at local slaughterhouses. All fetuses were serologically negative for Brucella spp. by using the Acidified Antigen Buffered test with fetal amniotic fluid. Prior to obtaining the CAM, the perimetrium was thoroughly decontaminated with iodinated alcohol, the uterus was then opened and CAM removed and placed in sterile medium containing antibiotics. Snapwell plates were used for culturing CAM explants. Sterile rings and detachable supports were positioned over the intercotyledonary portion of the CAM explants. Excess tissue was removed from CAM explants, which were placed in 6 well culture plates containing supplemented sterile culture medium and incubated at 37°C in a humidified atmosphere with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
Labeled cRNA was prepared from 0.5 ug RNA using the Two-Color Quick amp Iabelink kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
bovine trophoblastic cells [reference]
|
| Organism |
Bos taurus |
| Characteristics |
source tissue: intact pregnant bovine uteruses at the final third of gestation
tissue: Chorioallantoic membrane explant culture (CAM)
cell type: trophoblastic cells
infection: Non-infected
sample type: reference
|
| Treatment protocol |
The trophoblastic surface of the CAM explants was inoculated with 2.0 x 107 CFU, in a multiplicity of infection of approximately 1000 (MOI 1000:1). CAM explants were inoculated in triplicates with wild type, ΔvirB2, or ΔbtpB B. abortus 2308. Plates were incubated at 37°C with 5% CO2 for 4 h, the medium was then replaced with medium supplemented with 50 µg gentamicin/mL (Invitrogen, São Paulo, Brazil) to inactivate extracellular bacteria. Plates were maintained at 37°C with an atmosphere containing 5% CO2 for 1 h followed by washing three times with with PBS (phosphate buffered saline - pH 7.4) to eliminate the antibiotic. Uninfected CAM controls were inoculated with sterile medium and kept under the same conditions
|
| Growth protocol |
Seven intact pregnant bovine uteruses at the final third of gestation were obtained at local slaughterhouses. All fetuses were serologically negative for Brucella spp. by using the Acidified Antigen Buffered test with fetal amniotic fluid. Prior to obtaining the CAM, the perimetrium was thoroughly decontaminated with iodinated alcohol, the uterus was then opened and CAM removed and placed in sterile medium containing antibiotics. Snapwell plates were used for culturing CAM explants. Sterile rings and detachable supports were positioned over the intercotyledonary portion of the CAM explants. Excess tissue was removed from CAM explants, which were placed in 6 well culture plates containing supplemented sterile culture medium and incubated at 37°C in a humidified atmosphere with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
Labeled cRNA was prepared from 0.5 ug RNA using the Two-Color Quick amp Iabelink kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
825 ng of Cy3-labelled cRNA and 825 ng of Cy5-labelled cRNA (specific activity >10.0 pmol Cy3 or Cy5/ug cRNA) were mixed and fragmented at 60°C for 30 minutes in a reaction volume of 50 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 50 ull of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Bovine Oligo Microarrays design ID 023647 - GPL11648 (4x44K ) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Biological replicate 4 of 4. Chorioallantoic membrane explant culture (CAM) infected-wild type B. abortus 2308
Sample W9
|
| Data processing |
Agilent Feature Extraction Software (v 10.7.3.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Jun 04, 2014 |
| Last update date |
Sep 04, 2014 |
| Contact name |
Alex FIORINI Carvalho |
| E-mail(s) |
alexficar@gmail.com
|
| Phone |
5531982596868
|
| Organization name |
UFMG
|
| Department |
DMVP
|
| Lab |
AQUACEN
|
| Street address |
Avenida Antonio Carlos 6627
|
| City |
Belo Horizonte |
| State/province |
MG |
| ZIP/Postal code |
31010500 |
| Country |
Brazil |
| |
|
| Platform ID |
GPL11648 |
| Series (1) |
| GSE58216 |
Transcriptional profile of bovine chorioallantoic membrane explants in response to wild type, ΔvirB2 or ΔbtpB Brucella abortus |
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