|
| Status |
Public on Oct 11, 2019 |
| Title |
LNCaP radioresistant replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
Radioresistant LNCaP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: derived from LNCaP
phenotype: Radioresistant
|
| Treatment protocol |
Radioresistant sub-line was generated from original (parental) DU145 or LNCaP cell line by multiple X-ray irradiations, 4Gy/dose, 1 week recovery between doses. Prior to RNA isolation cells were stained with Aldefluor kit (Stem Cell Technologies) and sorted by FACS.
|
| Growth protocol |
Prostate cancer cell lines DU145 and LNCaP were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured according to the manufacturers recommendations in a humidified 37°C incubator supplemented with 5% CO2. DU145 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich) and LNCaP cells in RPMI-1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum (FBS, PAA Laboratories) and 1mM L-glutamine (Sigma-Aldrich).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with RNeasy mini kit (Qiagen) following the manufacturer's recommendations
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25ul of 2x GEx Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent-039494 SurePrint G3 Human GE v2 8x60K Microarray for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute at 37°C wit GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner G2505C using scan protocol Agilent G3_GX_1_color for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green)
|
| Description |
Gene expression profiling
|
| Data processing |
Quantile normalization was used to normalize the hybridization signals (gProcessedSignal). This was done separately for all DU145 and LNCaP samples to account for differences between DU145 and LNCaP.
|
| |
|
| Submission date |
Jul 18, 2019 |
| Last update date |
Oct 11, 2019 |
| Contact name |
Michael Seifert |
| Organization name |
TU Dresden
|
| Street address |
Fetscherstr. 74
|
| City |
Dresden |
| ZIP/Postal code |
01307 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17077 |
| Series (2) |
| GSE134499 |
Comparative analysis of gene expression profiles comparing radioresistant to radiosensitive prostate cancer cell lines |
| GSE134500 |
DNA copy number alteration analysis and gene expression profiles comparing radioresistant to radiosensitive prostate cancer cell lines |
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