|
| Status |
Public on Mar 27, 2012 |
| Title |
Peripheral mononuclear blood cells in patients with Takayasu's arteritis 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Peripheral blood cells from vasculitis patient 5
|
| Organism |
Homo sapiens |
| Characteristics |
gender: F
age (y): 54
disease state: Takayasu's vasculitis
cell type: peripheral blood cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNAs of TA patients' and normal volunteers' PBMCs were prepared by a standard method. Briefly, heparinized venous blood (10 mL) was mixed with an equal volume of 2% dextran/saline solution and incubated at room temperature for 30 min to precipitate the red blood cells. The PBMCs in the supernatant were then purified by density-gradient centrifugation on Percoll (density=1.064 g/mL). Total RNA was extracted from the PBMC pellets by adding guanidine-thiocyanate solution and the samples were used for acid guanidinium-phenol-chloroform extraction followed by ethanol precipitation and DNase I treatments for microarray analysis.
|
| 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 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 mononuclear blood cells from 17 healthy controls
|
| Organism |
Homo sapiens |
| Characteristics |
gender: 11 females and 6 males
age (y): avg. 42.1
disease state: healthy controls without any identified diseases
cell type: Peripheral mononuclear blood cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNAs of TA patients' and normal volunteers' PBMCs were prepared by a standard method. Briefly, heparinized venous blood (10 mL) was mixed with an equal volume of 2% dextran/saline solution and incubated at room temperature for 30 min to precipitate the red blood cells. The PBMCs in the supernatant were then purified by density-gradient centrifugation on Percoll (density=1.064 g/mL). Total RNA was extracted from the PBMC pellets by adding guanidine-thiocyanate solution and the samples were used for acid guanidinium-phenol-chloroform extraction followed by ethanol precipitation and DNase I treatments for microarray analysis.
|
| 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 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).
|
| |
|
| |
| 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 Takayasu's arteritis 5
|
| Data processing |
The image files were extracted using Agilent Feature Extraction software version 9.1.3.1 and LOWESS background subtraction and dye-normalization were used.
|
| |
|
| Submission date |
Nov 23, 2011 |
| Last update date |
Mar 28, 2012 |
| Contact name |
Daisuke Okuzaki |
| E-mail(s) |
dokuzaki@biken.osaka-u.ac.jp
|
| 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 (1) |
| GSE33910 |
Enhanced expression of Ficolin 1 is a specific gene marker of Takayasu's vasculitis |
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