|
| Status |
Public on Dec 10, 2012 |
| Title |
MBEZ |
| Sample type |
RNA |
| |
|
| Source name |
normal nasopharyngeal biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
disease state: normal control
|
| Biomaterial provider |
UMR542 Inserm-Université Paris Sud
|
| Extracted molecule |
total RNA |
| Extraction protocol |
8 micron cryosections were transferred onto PALM membrane slides (P.A.L.M. Microlaser Technologies) and air dried on ice for about 1 minute. Slides were immersed for 2 minutes in cold 75% ethanol, tapped dry and stained for 30 seconds in cold haematoxylin and eosin (9:1) containing 1% NucleoGuard (AmpTec, Hamburg). Excess stain was tapped off and slides were washed in cold nuclease-free water for 30 seconds, cold 75% ethanol for 1 minute, cold 100% ethanol for 1 minute then air dried. Cells for analysis were excised by laser microdissection and pressure catapulting using a PALM MicroBeam instrument and caught on PALM Adhesive Caps. A minimum of 200,000 µm2 of tissue was collected for each DNA or RNA extraction. RNA was extracted by adding 100µl of RLT buffer (Qiagen) supplemented with 1µl of N-carrier (AmpTec) and 1 µl of NucleoGuard followed by incubation at room temperature for 15 minutes. Extracted RNA was cleaned up using a Qiagen RNeasy mini kit, including the on-column DNase step as per the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
The eluted RNA was collected by ethanol precipitation in the presence of 1 µl P-carrier (AmpTec), washed twice with 80% ethanol and subjected to three rounds of amplification followed by biotin labelling using an ExpressArt TR Nano amplification kit (AmpTec) and an Affymetrix IVT labelling kit.
|
| |
|
| Hybridization protocol |
Biotinylated RNA was fragmented and hybridised to Affymetrix Human Genome U133Plus2 Arrays according to the Affymetrix protocol.
|
| Scan protocol |
All arrays were washed and stained on an Affymetrix FS450 fluidics station then scanned using an Affymetrix GeneChip 3000 7G scanner as per Affymetrix procedures.
|
| Data processing |
Expression array data were analysed with GCOS using the default settings except that the target signal was set to 100.
|
| |
|
| Submission date |
Dec 19, 2011 |
| Last update date |
Dec 10, 2012 |
| Contact name |
Wenbin Wei |
| E-mail(s) |
Wenbin.Wei2@durham.ac.uk
|
| Organization name |
Durham University
|
| Department |
Biosciences
|
| Street address |
Stockton Road
|
| City |
Durham |
| ZIP/Postal code |
DH1 3LE |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE34573 |
A Global View of the Oncogenic Landscape in Nasopharyngeal Carcinoma:An Integrated Analysis at the Genetic and Expression Levels |
|
