|
| Status |
Public on Nov 10, 2010 |
| Title |
Peripheral mononuclear blood cells in patients with vasculitis 4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Peripheral mononuclear blood cells from 16 healthy controls
|
| Organism |
Homo sapiens |
| Characteristics |
disease status: healthy controls without any identified diseases
gender: 9 females and 7 males
age: avg 38.2
|
| 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 |
Cy3
|
| 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 (pooled sample from 16 healthy control patients) 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 3-CTP (Cy3-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 A552nm for Cy3-CTP with Agilent 2100 Bioanalyzer (Agilent Technologies).
|
| |
|
| Channel 2 |
| Source name |
Peripheral blood cells from vasculitis patient 4
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
age: 41
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 |
Before hybridization, 825ng labeled cRNA of each product was fragmented and mixed with control targets and hybridization buffer according to the manufacturer's protocol (Agilent Technologies). Hybridizations were done for approximately 17h at 65°C. The slides were washed according to the manufacturer's manual, and scanning of microarrays was performed with 5µm resolution using a DNA microarray laser scanner (Agilent Technologies).
|
| Scan protocol |
The array was scanned using Agilent G2505B DNA microarray scanner.
|
| Description |
Genes expressed in peripheral blood mononuclear cells from patients with vasculitis.
|
| Data processing |
The image files were extracted using Agilent Feature Extraction software version 9.5.1 and LOWESS background subtraction and dye-normalization were used.
|
| |
|
| Submission date |
Jul 03, 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 |
GPL4133 |
| Series (2) |
| GSE16945 |
Comparative transcription profiling of peripheral blood mononuclear cells from vasculitis patients |
| GSE18316 |
Genopal: a novel platform for focused microarray analysis |
|
