|
| Status |
Public on Mar 10, 2014 |
| Title |
CF Male |
| Sample type |
RNA |
| |
|
| Source name |
endobronchial brushing
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: bronchial epithelium
gender: male
phenotype: cystic fibrosis
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The bronchial brush was immediately placed in 5 ml MEM+Glutamax supplemented with 10% FCS and 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA). Brushes were gently agitated to dislodge cells into the media, which was centrifuged at 300 × g for 5 minutes, and cell pellets were resuspended in 0.5 ml Tri Reagent (Sigma-Aldrich) before RNA extraction as per manufacturer’s protocol. The quality and the concentration of the RNA samples were monitored at absorbance ratios of A260/A280 and A260/A230 using a NanoDrop 8000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
|
| |
|
| Hybridization protocol |
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × blocking agent and 1 μl of 25 × fragmentation buffer, heated to 60 °C for 30 min, and diluted with 25 μl 2 × GE hybridization buffer. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the Human LncRNA Array v3.0 slide (8 x 60K, Arraystar). The slides were incubated for 17 hours at 65°C in an Agilent hybridization oven then washed, fixed and scanned.
|
| Scan protocol |
The slides were scanned using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Description |
lncRNA and mRNA expression
|
| Data processing |
The scanned were images were analysed using the Agilent Feature Extraction software version 11.0.1.1. Data normalization was carried out using Agilent GeneSpring GX. After quantile normalization of the raw data, LncRNAs and mRNAs that at least 1 out of 6 samples have flags in Present or Marginal were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs between two groups were identified through Volcano Plot filtering.
|
| |
|
| Submission date |
Feb 19, 2014 |
| Last update date |
Mar 10, 2014 |
| Contact name |
Paul Joseph McKiernan |
| E-mail(s) |
pauljmckiernan@rcsi.ie
|
| Organization name |
RCSI
|
| Department |
Medicine
|
| Lab |
Respiratory
|
| Street address |
Beaumont Hospital - ERC
|
| City |
Dublin |
| ZIP/Postal code |
09 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE55146 |
Aberrant expression of long noncoding RNA in vivo in the cystic fibrosis bronchial epithelium. |
|
