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Status |
Public on May 15, 2021 |
Title |
PNT1A_DHS_1e-8M_rep3 |
Sample type |
RNA |
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Source name |
PNT1A_48hr_1e-8M DHS
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Organism |
Homo sapiens |
Characteristics |
tissue origin: prostate Stage: non cancerous cell line: PNT1A cell type: prostate cell line treated with: 1e-8M DHS for 48hrs
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Treatment protocol |
PNT1A cells were treated with 1e-06 M (mid) or 1e-08 M (low) of plasticizers, or 0.4% DMSO (vehicle) for 48h at 37°C and 5% CO2
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Growth protocol |
PNT1A cells were grown at 37°C, 5% CO2 in RPMI 1640 supplemented with 0.025 M glucose, 2mM glutamine, 10mM Hepes, 1mM sodium pyruvate and 0.018 M sodium bicarbonate, 10% head inactivated fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 ug/mL of streptomycin
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was prepared using the RNEasy Plus Mini Kit, according to the manufacturer's protocol. RNA quality and quantification was obtained using the Bioanalyzer 2000 and the Nano 6000 RNA kit. Protein and other contaminants were assessed using the NanoDrop 2000.
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Label |
Cy3
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Label protocol |
25 ng of total RNA was used for labeling, according to Agilent's One color Low Input Quick Amp labeling kit protocol.
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Hybridization protocol |
600 ng 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 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K microarrays (G4851A) 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), dried briefly and put in Agilent Ozone-Barrier Slide holder.
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Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G2600D) using one color scan setting for 8x60k array slides
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Description |
Gene expression after 48hr incubation with DHS 1e-8M DHS_low_3
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Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters (protocol GE1_1105_Oct12 and Grid 028004_D_F_20120411) to obtain background subtracted and Processed Signal intensities. Please note that control probes were filtered out and only probes for which there was at least 4 samples with normalized signal of at least 10% higher to the background were included in the analysis.
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Submission date |
Mar 29, 2015 |
Last update date |
May 15, 2021 |
Contact name |
Claudia Lalancette |
E-mail(s) |
clalance@umich.edu
|
Organization name |
McGill University
|
Department |
Pharmacology and Therapeutics
|
Street address |
3655 Sir William Osler
|
City |
Montreal |
State/province |
QC |
ZIP/Postal code |
H3Y 1G6 |
Country |
Canada |
|
|
Platform ID |
GPL14550 |
Series (2) |
GSE67396 |
A Hightroughput Approach for Screening Potential Green Plasticizers Using A Human Prostate Cell Line [set1] |
GSE67399 |
A Hightroughput Approach for Screening Potential Green Plasticizers Using A Human Prostate Cell Line |
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