|
| Status |
Public on Feb 06, 2013 |
| Title |
NS_P-03 |
| Sample type |
RNA |
| |
|
| Source name |
normal skin
|
| Organism |
Homo sapiens |
| Characteristics |
patient: P-03
gender: male
age: 56
location: lower back
transplanted organ: kidney
immunosuppressive drugs: mycophenolate mofetil + prednison
sample type: normal unexposed epidernis
cell type: keratinocyte
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from SCC and AK samples that contained at least 70% tumor cells, as determined by haematoxylin and eosin stained frozen sections. From the sample of unexposed NS the epidermis was removed for further processing by cryosectioning parallel to the outer surface of the skin biopsy. RNA was extracted from frozen material using the RNeasy Fibrous Tissue kit (Qiagen), which included proteinase K treatment (10 min at 55˚C) of the lysed sample in RLT-buffer and on-column DNase treatment. RNA was quantified using a Nanodrop (NanoDrop technologies) and evaluated for degradation with a RNA 6000 Nano Labchip on the 2100 Bioanalyzer (Agilent Technologies)
|
| Label |
biotin
|
| Label protocol |
100 ng of total RNA was converted to cDNA and subsequently labeled cRNA using the Ambion Illumina TotalPrep RNA Amplification kit (Ambion) according to manufacturer’s instructions
|
| |
|
| Hybridization protocol |
The standard Illumina hybridization protocol was used. In brief, the samples were hybridized to the arrays at 58ºC overnight.
|
| Scan protocol |
The beadChips were scanned using the Illumina BeadArray Reader, using the standard Illumina scanning protocol
|
| Description |
4193399019_C
|
| Data processing |
Raw data was extracted from the BeadChip data files in Illumina’s BeadStudio Version 3.2 software using the gene expression module (v 3.2.7). Background subtracted data was further analyzed in R-based Bioconductor package, lumi (version 1.12.4). In lumi, the data was transformed (variance-stabilizing transformation (VST)) and normalized (robust spline normalization (RSN)), resulting in log-transformed normalized data. The R-package illuminaHumanv2.db (version 1.4.1) was used for annotation. The data were purged of genes that did not meet the detection limit (expression-detection P-value >0.01) and/or were not annotated. The limma R-package (version 3.2.3) was used to identify differentially expressed genes (DEGs) between SCC, AK and NS. Gene set enrichment analysis (GSEA) was performed with the significantly DEGs from the limma analysis using DAVID Bioinformatic Resources v6.7 (http://david.abcc.ncifcrf.gov). GSEA on the entire data set was performed using the parametric gene set enrichment analysis (PGSEA) R-package (version 1.14.0). To identify activation of transcription factors in AKs and SCCs, the DEGs from the limma analysis were investigated using the online analysis tool oPOSSUM.
Matrix normalized matrix shows VST-transformed, RSN-normalized data (used scripts from lumi package)
Matrix non-normalized: AVG_Signal: average signal for the probe; BEAD_STDERR: standard error of the beads; Avg_NBEADS: average number of beads for that probe; Detection Pval: detection p-value. All extracted from Beadstudio
|
| |
|
| Submission date |
Oct 05, 2011 |
| Last update date |
Feb 06, 2013 |
| Contact name |
Harry Vrieling |
| E-mail(s) |
h.vrieling@lumc.nl
|
| Organization name |
Leiden University Medical Center
|
| Department |
Toxicogenetics, S4-P
|
| Lab |
room T4-34
|
| Street address |
Einthovenweg 20
|
| City |
Leiden |
| ZIP/Postal code |
2333 ZC |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL6102 |
| Series (3) |
| GSE32628 |
Molecular profiling of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients |
| GSE32969 |
Genome-wide SNP analysis of cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients |
| GSE32979 |
Cutaneous squamous cell carcinomas and actinic keratoses from organ transplant recipients |
|
