|
| Status |
Public on Dec 01, 2012 |
| Title |
4616726055_B |
| Sample type |
RNA |
| |
|
| Source name |
Synovial biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
biopsy: normal control
|
| Extracted molecule |
total RNA |
| Extraction protocol |
biopsy collection: Fifteen knee synovial biopsy tissue samples consisting of six seronegative spondyloarthropy (SpA), two ankylosing spondylitis (AS), three osteoarthritis (OA) and four normal control biopsies were obtained from the Synovial Tissue Bank at the Repatriation General Hospital in Adelaide, South Australia with the appropriate ethical approval (Supplementary Table 1). All patients provided informed written consent.
RNA was extracted from the formaldehyde-fixed paraffin-embedded (FFPE) tissue blocks using the Arcturus Paradise© Plus Reagent System (Molecular Devices, Sunnyvale, CA) as per the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
200ng of RNA was used in the Illumina Whole-Genome DASL® (cDNA-mediated Annealing, Selection, Extension, and Ligation) Gene Expression Assay according to the Illumina protocol. This technique has been specifically developed for whole-genome expression profiling of degraded RNA samples from archived tissue biopsies. This protocol has been specifically developed for whole-genome expression profiling of degraded RNA samples from archived tissue biopsies. RNA is first converted to cDNA through a reverse transcription reaction with biotinylated primers. The biotinylated cDNA is then annealed to assay oligonucleotide probes specific for each of the 24000 cDNAs targeted by the array. The hybridized oligonucleotides are then extended and ligated in a second-strand cDNA synthesis forming a synthetic template that is transferred to a PCR reaction containing a fluorescently labelled primer. The labelled PCR product strand is then isolated and the fluorescent products were hybridised to Illumina Ref-8 Expression BeadChips and scanned.
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol
|
| Scan protocol |
Standard Illumina scanning protocol
|
| Description |
RNA extracted from FFPE biopsies
|
| Data processing |
Array data were processed using the Illumina BeadStudio software then the processed data were assessed for quality control and normalised in Lumi. Analysis of gene expression patterns was performed in BRB-ArrayTools. For quality control scanned images of the arrays were visually inspected for artefacts in Illumina BeadStudio followed by the graphical analysis of density plots in Lumi. The microarray data were transformed by variance stabilization transformation (VST) then normalized by robust spline normalization (RSN). Post-normalization quality control was carried out using density plots to ensure all samples had similar distribution and variance. To reduce background noise in the data analysis all probes that were not expressed in any of the samples, i.e. whose intensity was below background in all samples, were excluded from the analysis. This did not exclude probes for genes whose expression was either “switched-on” or “switched-off”.
|
| |
|
| Submission date |
Sep 20, 2012 |
| Last update date |
Dec 01, 2012 |
| Contact name |
Gethin Thomas |
| E-mail(s) |
gethin.thomas@uq.edu.au
|
| Phone |
+61 7 3176 2755
|
| Fax |
+61 7 3176 5946
|
| Organization name |
University of Queensland Diamantina Institute
|
| Street address |
Princess Alexandra Hospital
|
| City |
Woolloongabba |
| State/province |
QLD |
| ZIP/Postal code |
4102 |
| Country |
Australia |
| |
|
| Platform ID |
GPL6883 |
| Series (1) |
| GSE41038 |
Ankylosing spondylitis vs control synovial biopsies |
|
