|
| Status |
Public on Dec 31, 2015 |
| Title |
Primary prostate p53-/- Rb∆f/∆f 1 |
| Sample type |
RNA |
| |
|
| Source name |
Prostate, Cre
|
| Organism |
Mus musculus |
| Characteristics |
tissue: prostate
genotype: p53-/- Rb deltaf/deltaf
rb status: Deleted
pten status: Intact
|
| Treatment protocol |
Prostate cells were infected with adeno virus (lacZ) as a control or adeno virus (Cre) for deleting Rb overnight followed by changing of media. RNA was extracted after infection by 48 hours.
|
| Growth protocol |
Prostate cells were cultured in FBS lack Prostacult media supplemented with bFGF and bEGF
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy Mini Kit (#74106, Qiagen) according to the manufacturer’s instruction. RNA was quantified, and quality was monitored by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng of total RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions with some changes. 3rd Spike mix was made by diluted 2nd Spike-Mix 10 times, and 1.8 ul of 3rd Spike-Mix was added to samples. Cy3 labeled cRNA was synthesized at 40°C for 2 hours and purified by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Mouse Whole Genome Microarray 4x44K v2 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 with 37°C GE Wash buffer 2 (Agilent), then dried by raise it slowly from buffer 2.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green.
|
| Description |
Gene expression of p53-/- prostate cells in the depletion of Rb
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026655_D_F_20120201) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Entities having Detected flag across all samples were extracted.
|
| |
|
| Submission date |
May 14, 2015 |
| Last update date |
Dec 31, 2015 |
| Contact name |
Takumi Nishiuchi |
| E-mail(s) |
tnish9@staff.kanazawa-u.ac.jp
|
| Organization name |
Kanazawa University
|
| Street address |
13-1 Takaramachi
|
| City |
Kanazawa |
| ZIP/Postal code |
920-0934 |
| Country |
Japan |
| |
|
| Platform ID |
GPL11202 |
| Series (2) |
| GSE68903 |
Development of gene sets after deleting Rb in p53 null background primary prostate cells [Rb only] |
| GSE68905 |
Development of gene sets after deleting Rb or Rb and Pten in p53 null background primary prostate cells |
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