|
| Status |
Public on Jan 19, 2022 |
| Title |
Peripheral blood monocytes from COPD Smokers replicate 3 |
| Sample type |
RNA |
| |
|
| Source name |
Peripheral blood monocytes
|
| Organism |
Homo sapiens |
| Characteristics |
gender: MALE
cells: peripheral blood monocytes
age: 69
phenotype: COPD EX Smokers
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral blood from Control Smokers, Control Never smokers, COPD Smokers and COPD Ex-smokers was isolated followed by isolation of Peripheral Blood mononuclear cells (PBMC's). CD14+ monocytes were isolated from PBMC's using CD14 microbeads following manufacturer's instructions. RNA was isolated from peripheral blood CD14+ monocytes using DirectZol RNA extraction kit following manufacturer's recommendations.This protocol includes RNA isolation and an on-column DNase digestion. RNA was quantified using a Nanodrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng total RNA using the One-Color Low Input quickAmp Labeling kit (Agilent) according to manufacturer's instructions, followed by RNeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 µg of Cy3-labelled cRNA (specific activity >6 pmol Cy3/ µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 µl containing 1X Agilent fragmentation buffer and 2X Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 µl of 2X Agilent hybridization buffer was added to the fragmentation mixture and hybridized to 4 X 44k Agilent Whole Human Genome Oligo Microarrays for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash Buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one colour scan setting for 4 X 44k array slides (Scan Area 61X21.6 mm, Scan resolution 5 µm, Dye channel was set to Green, and Green PMT was set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1 and Design Id: 014850) to obtain background subtracted and spatially detrended Processed Signal Intensities. Features were flagged in Feature Extraction and Non-uniforn Features and outliers were excluded.
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| |
|
| Submission date |
Mar 06, 2020 |
| Last update date |
Jan 19, 2022 |
| Contact name |
Anjana Talwar |
| E-mail(s) |
anjanatalwar@gmail.com
|
| Organization name |
All India Institute of Medical Sciences
|
| Department |
Physiology
|
| Lab |
Respiratory Research Lab
|
| Street address |
Ansari Nagar
|
| City |
New delhi |
| State/province |
Delhi |
| ZIP/Postal code |
110029 |
| Country |
India |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE146560 |
Homo sapiens whole genome expression microarray of peripheral blood monocytes obtained from Control Smokers, Control Never Smokers, COPD Smokers and COPD ExSmokers. |
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