|
| Status |
Public on Nov 30, 2009 |
| Title |
Peripheral blood mononuclear cells (PBMC)- Healthy subject NORM5 |
| Sample type |
RNA |
| |
|
| Source name |
PBMC NORM5
|
| Organism |
Homo sapiens |
| Characteristics |
healthy subject: NORM5
group: Training
|
| Biomaterial provider |
Department of Emergency and Organ transplantation, Nephrology Unit, University of Bari
|
| Treatment protocol |
PBMC were immediately isolated from 20 ml of whole blood by Ficoll–Hypaque (Flow Laboratories, Irvine, UK) density gradient centrifugation. Isolated cells were stored at –80°C in FBS+10% DMSO until use.
|
| Growth protocol |
Before RNA extraction, cells were thaw, counted and their viability determined by Trypan Blue exclusion (>90% PBMC were viable). A minimum of 5 milion cryopreserved PBMC were centrifuged, and immediately lysed for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by RNeasy mini kit (QIAGEN Inc., Valencia, CA) according to the manufacturer’s instructions and resuspended in DEPC-treated Rnase-free distilled water. RNA concentration was determined by NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE) and its integrity was assessed by electrophoresis using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA).
|
| Label |
Biotin
|
| Label protocol |
The One-Cycle cDNA Synthesis Kit (Affymetrix) was used. Starting from 1.5 micrograms of total RNA we performed the in vitro transcription (IVT) reaction for complementary RNA (cRNA) amplification and biotin labeling, according to the manufacturer’s instructions. The biotinylated cRNA targets were then cleaned up, fragmented, and hybridized to GeneChip brand expression arrays.
|
| |
|
| Hybridization protocol |
Affymetrix GeneChips Human Genome U133 Array Set HG-U133A (Affymetrix) were hybridizated with 15 micrograms of fragmented cRNA, according to manufacturer’s instructions
|
| Scan protocol |
We used the default settings of GCOS software to scan the chips
|
| Description |
We used the default settings of GCOS software to scan the chips
|
| Data processing |
We used the default settings of Affymetrix Microarray Suite software version 5 (MAS 5.0; Affymetrix) to calculate scaled gene expression values.
|
| |
|
| Submission date |
Mar 03, 2009 |
| Last update date |
Nov 30, 2009 |
| Contact name |
Paola Pontrelli |
| E-mail(s) |
paola.pontrelli@uniba.it
|
| Phone |
+390805478868
|
| Organization name |
University of Bari
|
| Department |
Dept. of Precision and Regenerative Medicine and Ionian Area (DIMEPRE-J)
|
| Lab |
Nephrology Unit
|
| Street address |
Piazza G.Cesare 11
|
| City |
Bari |
| ZIP/Postal code |
70124 |
| Country |
Italy |
| |
|
| Platform ID |
GPL96 |
| Series (1) |
| GSE15072 |
Mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease |
|
