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Status |
Public on Jul 10, 2012 |
Title |
LNCaP-FASN-RNAi cells replicate2 |
Sample type |
RNA |
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Source name |
LNCaP-FASN-RNAi cells_replicate2
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Organism |
Homo sapiens |
Characteristics |
cell line: LNCaP transfection: FASN-RNAi
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Treatment protocol |
Cells (1 × 10^4) were plated in 100 ul growth medium per well on 96-well plates and incubated overnight. The medium was then changed to growth medium containing hexadimethrine bromide (Sigma) at a final concentration of 8 ug/ml to enhance the transduction efficiency. Lentiviral particles (5-50 ul, 0.5-5 multiplicity of infection) were added and the plates were incubated overnight. Thereafter, we selected stable transductants expressing the shRNAs with puromycin.
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Growth protocol |
Cells were incubated in a humidified atmosphere of 5% CO2 in air at 37°C. RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and antibiotics was used for growth medium. Exponentially growing cells were used for the experiments. Cells were trypsinized, and the number of viable cells was counted by the trypan blue dye-exclusion method.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA isolation was performed with a Micro-to-Midi total RNA purification system (Invitrogen). The integrity of total RNAs was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies).
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Label |
Cy3
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Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1ug Total RNA using the Low Input Quick Amp Labeling Kit(Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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Hybridization protocol |
0.6 ug of Cy3-labelled 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 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 immediately by brief centrifugation.
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Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).
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Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
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Submission date |
Jul 09, 2012 |
Last update date |
Aug 06, 2012 |
Contact name |
Yukie Yoshii |
E-mail(s) |
yoshii.yukie@qst.go.jp
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Phone |
043-206-3429
|
Organization name |
National Institute of Radiological Sciences
|
Street address |
Anagawa 4-9-1, Inage
|
City |
Chiba |
ZIP/Postal code |
263-8555 |
Country |
Japan |
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Platform ID |
GPL13607 |
Series (1) |
GSE39183 |
Characterization of FASN knockdown LNCaP cells |
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