|
| Status |
Public on Apr 09, 2011 |
| Title |
Syngeneic_03 |
| Sample type |
RNA |
| |
|
| Source name |
Syngeneic
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: Kidney transplant
transplantation: Syngeneic
day post transplantation: 100
|
| Treatment protocol |
Lewis 1W (LEW.1W, haplotype RT1u) and Lewis 1A (LEW.1A, haplotype RT1a) rats (Centre d’Elevage Janvier, Le Genest-Saint-Isle, France) share the same genetic background but are MHC I and II mismatched. Heterotopic transplantations of 8 to 12 weeks old LEW.1W kidneys on LEW.1A animals were carried out according to the technique described by Ono & Lindsey. For syngeneic transplantations LEW.1A kidneys on LEW.1A recipients were achieved. Live-sustaining allotransplantations were performed aseptically and a binephrectomy was performed 7 days after transplantation as previously described. Blood urea and creatinine and urine protein/creatinine ratios were measured throughout the posttransplant period. Blood urea < 8 mM and blood creatinine < 40 mM were considered as normal. Transplant tolerance induction was obtained by treating rats with anti-donor anti-class II antibodies.
|
| Growth protocol |
Rats were maintained in an SPF-free animal facility according to the institutional guidelines of the “Institut National de la Santé et de la Recherche Médicale” (INSERM).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Organs were harvested at day 100 post transplantation. Sections of the kidney transplant were snap frozen in liquid nitrogen for RNA extraction. Total RNA from rat kidney transplants was prepared using the TRIzol (Invitrogen Life Technologies) extraction method. Ten micrograms of total RNA was cleaned up using RNeasy columns (Qiagen). RNA quantity and quality were determined using a NanoDrop spectrophotometer and an Agilent 2100 bioanalyzer. A rRNA 28S/18S ratio of 1.0 ± 0.1 was considered as acceptable for RNA amplification.
|
| Label |
Digoxigenin-UTP
|
| Label protocol |
The Applied Biosystems rat genome survey microarray (part no. 4337467) contained 26,857 60-mer oligonucleotide probes representing 27,088 individual rat genes. An additional 3400 control spots were present on the chip to cover various steps in the hybridization process. Digoxigenin-UTP labeled cRNA was generated and amplified from 0.5 µg of total RNA from each sample using an Applied Biosystems NanoAmp chemiluminescent reverse transcriptase-in vitro transcription (RT-IVT) labeling kit (part no. 4365715).
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| |
|
| Hybridization protocol |
Array hybridization was performed for 16 h at 55°C. An additional 3400 control spots were present on the chip to cover various steps in the hybridization process.
|
| Scan protocol |
Chemiluminescence detection, image acquisition, and analysis were performed using the Applied Biosystems chemiluminescence detection kit (part no. 436875D), analyzer (part no. 4338036) and version 1.1 analyzer software (part no. 4336391) according to the manufacturer’s protocol.
|
| Description |
Syngeneic
|
| Data processing |
Microarray raw data were analyzed by the R language and environment for statistical computing and graphics. Genes were identified using the Panther Protein Classification System Probe ID database.
|
| |
|
| Submission date |
Apr 08, 2011 |
| Last update date |
Apr 09, 2011 |
| Contact name |
Romain Vuillefroy de Silly |
| Organization name |
INSERM UMR643
|
| Street address |
30 Bvd Jean Monnet
|
| City |
Nantes |
| ZIP/Postal code |
44093 |
| Country |
France |
| |
|
| Platform ID |
GPL2996 |
| Series (1) |
| GSE28474 |
Comparison between syngeneic kidney transplant and a model of allogeneic kidney transplant tolerance induced by anti-classII regimen in the rat |
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