|
| Status |
Public on Apr 01, 2013 |
| Title |
PBMC_2_48 |
| Sample type |
RNA |
| |
|
| Source name |
Recipient 2; 4 Year; Tolerant
|
| Organism |
Homo sapiens |
| Characteristics |
recipient: 2
tissue: Peripheral Blood
time-point: 4 Year
induction: Alemtuzumab
tolerance: Tolerant
|
| Treatment protocol |
N/A
|
| Growth protocol |
N/A
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA Extraction from PAXgene Blood Tubes: Total RNA including small and miRNA is extracted with the PreAnalytiX PAXgene Blood miRNA kit ( QIAGEN). A semi automated method using the QIAcube (QIAGEN) is used following manufacturer’s instructions. The quantity and quality of the RNA is assessed on a nanodrop and Agilent Bioanalyzer. miRNAeasy kit PN 217004 (Qiagen)
|
| Label |
SAPE
|
| Label protocol |
The cDNA generated from the WT Expression Kit is fragmented and biotin labeled with the Affymetrix GeneChip WT Terminal Labeling Kit ( Affymetrix). The cDNA is fragmented with a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonucleaseI (APE I). The fragmented cDNA is then labeled with terminal deoxynucleotidyl transferase (TdT) and a biotin reagent.
|
| |
|
| Hybridization protocol |
The Gene Titan Hybridization, Wash and Stain Kit for WT Array Plates (Affymetrix) is used to hybridize 3ug of the labeled cDNA to a multi peg Gene 1.1 ST Array Plate. The hybridization cocktail also contains Control Oligo B2 and 20X Eukaryotic Hybridization Controls as recommended by Affymetrix. Hybridization is carried out for 17 hours at 48C in the Gene Titan MC (Multichannel) Instrument ( Affymetrix). After hybridization the peg array plate is automatically washed and stained and moved to the scanner.
|
| Scan protocol |
The peg array plate is automatically moved to the Xenon laser scanner of the Titan MC instrument and scanned at default settings recommended for the peg array.
|
| Description |
4 Year_TOL
|
| Data processing |
Probes to Import: Interrogating Probes and Control Probes, Probe filtering: skip, Algorithm: RMA, Background Correction: RMA Background Correction, Normalization: Quantile Normalization, Log Probes using Base: 2, Probeset Summarization: Median Polish
probe group file: HuGene-1_1-st-v1.r4.pgf
meta-probeset file: HuGene-1_1-st-v1.r4.mps
|
| |
|
| Submission date |
Mar 28, 2013 |
| Last update date |
Apr 01, 2013 |
| Contact name |
Sunil Kurian |
| E-mail(s) |
smkurian@scripps.edu
|
| Phone |
858-784-7759
|
| Organization name |
The Scripps Research Institute
|
| Department |
Molecular and Experimental Medicine
|
| Street address |
10550 N Torrey Pines Road
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL11532 |
| Series (1) |
| GSE45593 |
GENOMICS TO IDENTIFY HLA IDENTICAL RENAL TRANSPLANT TOLERANCE SIGNATURES |
|
