|
| Status |
Public on Jan 11, 2014 |
| Title |
Ad.Null 48h versus Ad.p75NTR 48h Replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
HUVEC infected by adenoviral vector Ad-rad66 (empty vector, also called Null)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HUVEC
|
| Treatment protocol |
To obtain p75 over-expressing cells, HUVEC were infected by adenoviral vectors carrying human p75NTR sequence (Ad. p75NTR) or by empty vector (Ad-rad66).
|
| Growth protocol |
HUVEC were grown in EGM-2 (Lonza) containing 2% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol (Invitrogen) extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Hy3
|
| Label protocol |
2.5 ug total RNA was labeled with Hy3, Hy5 resepctively according to the manufacturer's protocol (miRCURY LNA microRNA Array Power labeling kit, EXIQON).
|
| |
|
| Channel 2 |
| Source name |
HUVEC infected by adenoviral vectors carrying human p75NTR (Ad. p75NTR)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HUVEC
|
| Treatment protocol |
To obtain p75 over-expressing cells, HUVEC were infected by adenoviral vectors carrying human p75NTR sequence (Ad. p75NTR) or by empty vector (Ad-rad66).
|
| Growth protocol |
HUVEC were grown in EGM-2 (Lonza) containing 2% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol (Invitrogen) extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
Hy5
|
| Label protocol |
2.5 ug total RNA was labeled with Hy3, Hy5 resepctively according to the manufacturer's protocol (miRCURY LNA microRNA Array Power labeling kit, EXIQON).
|
| |
|
| |
| Hybridization protocol |
2-color hybridization was performed using miRCURY LNA microRNA Arrays (v.10.0, EXIQON). 2.5ug of each labeled RNA were co-hybridized at 53°C for 16 hours. Hybridization and washing steps were performed on the HS 400 PRO hybridization station (Tecan).
|
| Scan protocol |
MicroRNA arrays were scanned using the GenePix 4100A microarray scanner and the GenePix Pro 6.0.1.25 software (Molecular Devices).
|
| Description |
microRNA expression data from HUVEC over-expressing p75
|
| Data processing |
The obtained signal intensities were analyzed by using the limma package (Bioconductor 2.5 on R 2.10 ). Background correction (normexp, cutoff=10), within (global LOESS) -and between (scale method) array normalization was performed. Differential expression and statistical significance were assessed for each miRNA species across all 4 arrays using linear model fit and empirical Bayes method (lmFit, eBayes), taking into account the dye-swap. Features showing intensities with a signal-to-noise ration (SNR) lower than 3 were considered as bad quality spot. The calling of a miRNA failed if two or more of the four replicates present per miRNA species were bad quality spots and if this occurred on more than 3 arrays.
Log2FC: Normalized log2 ratio (Hy5/Hy3). P value adjusted for False Discovery Rate (Benjamini and Hochberg's method).
|
| |
|
| Submission date |
Jan 08, 2014 |
| Last update date |
Jan 11, 2014 |
| Contact name |
Andrea Caporali |
| E-mail(s) |
a.caporali@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Street address |
47 Litlte France Crescent
|
| City |
EDINBURGH |
| ZIP/Postal code |
EH16 4TJ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL7722 |
| Series (1) |
| GSE53899 |
Effect of neurotrophin receptor p75 (p75NTR) over-expression on the miRNAome of human umbilical vein endothelial cells |
|
