|
| Status |
Public on Jan 04, 2017 |
| Title |
Severe_Central_Patient_40 |
| Sample type |
RNA |
| |
|
| Source name |
Epithelial brushings from the central airways of a severe asthmatic
|
| Organism |
Homo sapiens |
| Characteristics |
patient id: 40
diagnosis: Severe Asthmatic
Sex: M
age: 33
date of hybridization: 2012-05-09
cell type: Central airway epithelium
|
| Growth protocol |
Epithelial brushings were collected at fibre optic bronchoscopy under local anaesthesia using disposable, sheathed bronchial brushes with 4 brushes from the central and 4 fromperipheral airways. Brushings were placed separately in sterile universal tubes with 5 ml PBS and 5 ml of RPMI medium (with 1% Pen/strep, 1% L-glutamine and 20% FBS) was added and the tubes spun at 1000 rpm for 5 minutes to pellet the cells. The medium was discarded and 1 ml of RNA later Reagent was added to the cell pellet in each universal tube, followed by pipetting up and down to solubilise the cells and incubation at room temperature for 5 minutes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from the samples using the Qiagen miRNeasy Kit. RNA quality was assessed using a Bioanalyzer 2100.
|
| Label |
biotin
|
| Label protocol |
Not provided.
|
| |
|
| Hybridization protocol |
Samples were hybridized to Affymetrix HG U133 plus 2.0 beadchips.
|
| Scan protocol |
Not provided.
|
| Description |
Epithelial brushings gene expression data from the central airways of a severe asthmatic
|
| Data processing |
Raw microarray gene expression data was normalized using GCRMA in R and subjected to several quality control procedures. Batch effects based on the date of microarray hybridization were removed. Differentially expressed genes were identified using a linear modelling approach in the limma package in R and only genes with FDR corrected p-values < 0.05 for multiple testing (using the Benjamini-Hochberg method) were considered significant.
For RT-qPCR, RNA was isolated and reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit with random hexamers. RT-qPCR was performed for each gene target in duplicate using Taqman Universal PCR Master Mix, No AmpErase UNG on 7900HT Fast Real-Time PCR System. GAPDH was used as a normalizer to obtain fold changes and t-tests were performed to determine significance. Thermal cycling conditions were 95˚C for 10 minutes followed by 40 cycles of 95˚C for 15 seconds and 60˚C for 1 minute.
|
| |
|
| Submission date |
Jan 13, 2015 |
| Last update date |
Jan 04, 2017 |
| Contact name |
Akul Singhania |
| E-mail(s) |
[email protected]
|
| Phone |
+442037963319
|
| Organization name |
The Francis Crick Institute
|
| Street address |
1 Midland Road
|
| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE64913 |
Altered epithelial gene expression in peripheral airways of severe asthma |
|
