|
| Status |
Public on Nov 29, 2011 |
| Title |
C17orf62_KD |
| Sample type |
RNA |
| |
|
| Source name |
HUVEC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: HUVEC
sirna knocked down: C17orf62 siRNA knocked down
|
| Treatment protocol |
HUVEC pools consisting of 10 biological isolates were transfected with siRNA smartpools from Dharmacon Inc. (Lafayette, Colorado, USA) using the siFectamine transfection reagent (ICVEC, London, UK) used according to the manufacturer’s instructions. Transfection was carried out when HUVEC pools were 60-80% confluent, with siRNA smartpools at a final concentration of 100nm per treatment and RNA was extracted 24hrs after transfection.
|
| Growth protocol |
HUVECs were isolated from umbilical cords by collagenase digestion and cultured at 37ºC/5%CO2 in basal culture medium supplemented with a proprietary mixture of heparin, hydrocortisone, epidermal growth factor, 2% foetal calf serum (FCS; EGM-2, Cambrex, Workingham, UK).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell cultures were washed with 1 x PBS and lysed using TRIzol® reagent. Total RNA was extracted using phenol chloroform, with the precipitated RNA washed in 70% ethanol and resuspended in Rnase free water. RNA concentration and quality was assess by spectrophotometer and Agilent BioAnalyser.
|
| Label |
biotin
|
| Label protocol |
Biotin-labelled complex cRNAs were prepared to according to the manufacturer's protocols (Applied Microarrays, formally supplied by GE Healthcare)
|
| |
|
| Hybridization protocol |
10 µg biotin incorporated cRNA was fragmented and then denatured. The denatured cRNA was slowly loaded into the bioarray flex chamber without the introduction of air bubbles. All samples were loaded within 30 minutes of cRNA denaturation. The sealed bioarrays were left to hybridise in a 37°C shaker-incubator for 18 hours. The slides were washed and placed in a solution of Cy5-streptavidin for 30 minutes, before stringent washes were used to remove any unbound dye.
|
| Scan protocol |
The arrays were scanned with Agilnet DNA Microarray Scanner model G2565BA.
|
| Description |
HUVEC_KD022
|
| Data processing |
The scanned images were quantified with CodeLink Expression Array Software (Applied Microarrays, formally supplied by GE Healthcare), where the intensity of each spot was divided by the spot’s signal median.
|
| |
|
| Submission date |
Mar 09, 2011 |
| Last update date |
Nov 29, 2011 |
| Contact name |
Hiromitsu Araki |
| E-mail(s) |
araki.hiromitsu.596@m.kyushu-u.ac.jp
|
| Organization name |
Kyushu University
|
| Street address |
744 Motooka Nishi-Ku
|
| City |
Fukuoka |
| ZIP/Postal code |
819-0395 |
| Country |
Japan |
| |
|
| Platform ID |
GPL4044 |
| Series (2) |
| GSE27869 |
Gene expression profiles of 400 siRNA knocked down on HUVEC |
| GSE27871 |
Network inference tools for the human transcriptome |
|
