|
| Status |
Public on Nov 10, 2010 |
| Title |
PBMCs from vasculitis patients 9 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral blood cells from vasculitis patient 9
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
age: 60
disease status: vasculitis
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral blood was collected directly into PAXGene tubes (Qiagen, Valencia, CA) and stored at -80℃ until extraction. Total RNA from mononuclear cells was extracted using PAXGene Blood RNA kit with the optimal on-column DNase digestion according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 500ng of total RNA (derived from vasculitis patients 1 with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled antisense cRNA was purified with RNeasy columns (Qiagen) according to manufacturer's protocols and resuspended in water. The purified products were quantified at A650nm for Cy5-CTP with Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| |
|
| Hybridization protocol |
Briefly, 1125 ng labeled cRNA was fragmented according to the manufacturer's protocol (Agilent Technologies), was suspended in a hybridization buffer containing 0.12 M Tris-HCl (pH 7.5), 0.12 M NaCl, and 0.05% Tween 20. The hybridization mixture was heated to 94°C for 2 min, then cooled to 37°C and injected into a hybridization chamber (Mitsubishi Rayon). Hybridization was carried out at 65°C for 16 hr. Microarrays were washed by soaking twice in 0.12 M Tris-HCl (pH 7.5), 0.12 M NaCl, 0.05% and Tween 20 at 65°C for 20 min, then once in 0.12 M Tris-HCl (pH 7.5), 0.12 M NaCl at 65°C for 10 min.
|
| Scan protocol |
Microarray was scanned and the image was captured using a cooled charge-coupled device (CCD)-type Microarray Image Analyzer equipped with multibeam excitation technology (Yokogawa Electric Co., Tokyo, Japan).
|
| Description |
Genes expressed in peripheral blood mononuclear cells from patients with vasculitis.
|
| Data processing |
Data analysis with background subtraction and normalization to alpha-tubulin intensity was conducted using the GeneSpring GX software version 10.0.2, according to the manufacturer’s protocol (Agilent Technologies).
|
| |
|
| Submission date |
Sep 24, 2009 |
| Last update date |
Nov 10, 2010 |
| Contact name |
Daisuke Okuzaki |
| E-mail(s) |
[email protected]
|
| Phone |
+81-6-6879-4935
|
| Organization name |
Osaka univ.
|
| Department |
Immunology Frontier Research Center
|
| Lab |
Human Immunology （Single Cell Genomics)
|
| Street address |
Yamadaoka 3-1
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL9283 |
| Series (2) |
| GSE18247 |
Focused transcription profiling of peripheral blood mononuclear cells from vasculitis patients |
| GSE18316 |
Genopal: a novel platform for focused microarray analysis |
|
