|
| Status |
Public on Aug 02, 2011 |
| Title |
Primary_9 |
| Sample type |
RNA |
| |
|
| Source name |
Malignant melanoma
|
| Organism |
Homo sapiens |
| Characteristics |
replicate samples: Unique sample
study type: Study 2
sample type: Primary
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cores of tissue were taken using a tissue microarray core. These were de-waxed using xylene and two changes of absolute ethanol. RNA was extracted using the column based technique Roche High Pure Paraffin RNA kit and treated with Dnase I according to the kit protocol. Quality control procedures included running samples on the ND-1000 spectrophotometer, the Agilent 2100 Bioanalyzer and performing quantitaive RT-PCR of RPL13a.
|
| Label |
Cy3 and Cy5
|
| Label protocol |
200ng total RNA was converted into cDNA using biotinylated random hexamers and oligo-deoxythymidine 18 primers, and Illumina-supplied reagents according to manufacturer’s instructions. Pairs of query oligos were then annealed to complementary sequences (~50 bases) flanking the specific cDNA target site. The biotinylated cDNA was then bound to streptavidin-conjugated paramagnetic particles and mis-hybridised and non-hybridised oligos were washed away. Hybridised oligos were then extended and ligated to generate amplifiable templates.
|
| |
|
| Hybridization protocol |
A PCR reaction was then performed with Cy3 and Cy5 labelled universal PCR primers and the double stranded PCR products were isolated and hybridised at 45°C for 18 hours to the Illumina DASL 502 gene Human Cancer Panel.
|
| Scan protocol |
After hybridisation the arrays were scanned using the BeadArray™ scanner and analysed by BeadStudio v3 software (Illumina, USA).
|
| Description |
4360118026_R008_C003
|
| Data processing |
The data were normalized using Beadstudio software (Illumina, USA) before exporting to STATA version 10 for statistical analyses. The normalisation methods used were background correction, cubic spline smoothing and plate scaling. Studies were normalized separately before samples were compared across studies. The mean gene expression across 3 probes targeting each gene was used for analysis.
|
| |
|
| Submission date |
Aug 02, 2011 |
| Last update date |
Aug 02, 2011 |
| Contact name |
Rosalyn Jewell |
| E-mail(s) |
r.a.jewell@leeds.ac.uk
|
| Organization name |
University of Leeds
|
| Department |
Section of Epidemiology and Biostatistics
|
| Street address |
Leeds Cancer Research UK Centre, LIMM, Cancer Genetics Building, St James;s University Hospital, Beckett Street
|
| City |
Leeds |
| ZIP/Postal code |
LS9 7TF |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11417 |
| Series (1) |
| GSE31139 |
Identification of differentially expressed genes in matched primary and metastatic melanoma tumor pairs |
|
