|
| Status |
Public on Sep 12, 2012 |
| Title |
RPE1_hTERT_Chr5Chr12_R3 |
| Sample type |
RNA |
| |
|
| Source name |
RPE-1 cell line, triploid for Chr5 and Chr12, replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: RPE-1
multisomy/variation: 3x Chr5 3x Chr12
|
| Treatment protocol |
no treatment
|
| Growth protocol |
Both HCT116 and RPE-1 cell lines were cultured in DMEM (supplemented with 10% FBS) at 5% CO2, 37°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mRNA was purified using the Qiagen mRNeasy mini kit.
|
| Label |
Cy3
|
| Label protocol |
500 ng (HCT116) or 100 ng (RPE-1) total RNA per sample were introduced into an RT-IVT reaction. Prior to RT-IVT, the total RNA samples were spiked with in-vitro synthesized polyadenylated transcripts (One-Color RNA Spike-In Mix, Agilent Technologies) which serve as an internal labeling control for linearity, sensitivity and accuracy. The spiked total RNA was reverse transcribed into cDNA and then converted into labeled cRNA by in-vitro transcription (HCT116: Quick-Amp Labeling Kit One-Color, Agilent Technologies; RPE-1: Low Input Quick-Amp Labeling Kit One-Color, Agilent Technologies) incorporating Cyanine-3-CTP according to the manufacturer´s instructions.
|
| |
|
| Hybridization protocol |
Following cRNA clean-up and quantification (NanoDrop ND-1000 Spectrophotometer, peqlab) 1.65 µg (HCT116) or 0.60 µg (RPE-1) of each Cyanin-3-labeled cRNA sample was fragmented and prepared for One-Color based hybridization (Gene Expression Hybridization Kit, Agilent). Each cRNA sample was hybridized at 65 °C for 17 hrs on separate Whole Human Genome Oligo Microarrays (4x44K format) (for HCT116) or SurePrint G3 Human Gene Expression Arrays (8x60K).
|
| Scan protocol |
Fluorescent signal intensities were detected with Scan Control 8.1.3. Software (Agilent Technologies) (HCT116) or Scan Control A.8.4.1 Software (Agilent Technologie) (RPE-1) on the Agilent DNA Microarray Scanner.
|
| Description |
Gene expression of RPE-1 cell lines incl. 3x Chr5 and 3x Chr12
|
| Data processing |
Signal intensities were extracted from the image using Feature Extraction 10.5.1 Software (Agilent Technologies) and the design file 014850_D_F_20090416.xml (HCT116) or 10.7.3.1 Software and the design file 028004_D_F_20120130.xml (RPE-1). Data was background subtracted; log2 ratios calculated and normalized. Medians of replicates were calculated and a ratio of the aneuploid cell line (median) vs the corresponding WT cell line (median) was used for further analysis (indicated in the column header).
|
| |
|
| Submission date |
Jul 31, 2012 |
| Last update date |
Sep 13, 2012 |
| Contact name |
Gabriele Stoehr |
| Organization name |
MPI of Biochemistry
|
| Department |
Proteomics ans Signaltransduction
|
| Street address |
Am Klopferspitz 18
|
| City |
Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE39768 |
Global analysis of genome, transcriptome and proteome reveals the response to aneuploidy in human cells |
|
