|
| Status |
Public on Jan 02, 2018 |
| Title |
HNE2_TP53 transfection_12 hours |
| Sample type |
RNA |
| |
|
| Source name |
HNE2_TP53 transfection_12 hours
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: nasopharyngeal carcinoma
gender: male
cell line: HNE2
transfection: TP53 for 12 hours
|
| Treatment protocol |
HNE2 were transfected with pCMV-p53 plasmid (Clontech Laboratories Inc.) using Lipofectamine 2000 according to the manufacturer’s suggested protocol.
|
| Growth protocol |
Human nasopharyngeal carcinoma (NPC) HNE2 cells were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS). Cells were grown at 37°C in a humidified 5% CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted by mirVanaTM RNA Isolation Kit (Applied Biosystem p/n AM1556 ) following the manufacturer's instructions
|
| Label |
cy3
|
| Label protocol |
0.1ug total RNA were dephosphorylated, denaturated and then labeled with Cyanine-3-CTP（Cy3）using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions,then followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1.
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| |
|
| 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 25x Agilent fragmentation buffer and 10x Agient 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-046065 Mouse miRNA Microarray V19.0 8x60K(Agilent) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
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| Description |
Gene expression after 12hr TP53 transfected HNE2 cell
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities as the raw data. Raw data were normalized in quantile algorithm with Genespring 12.0(Agilent). Probe that at least 100.0 percent of samples in any 1 condition out of 2 conditions have flags in Detected were maintained.
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| |
|
| Submission date |
Jan 02, 2015 |
| Last update date |
Jan 02, 2018 |
| Contact name |
Wei Xiong |
| E-mail(s) |
xiongwei@csu.edu.cn
|
| Organization name |
Central South University
|
| Department |
Cancer Research Institute
|
| Street address |
110 Xiangya Road
|
| City |
Changsha |
| State/province |
Hunan |
| ZIP/Postal code |
410078 |
| Country |
China |
| |
|
| Platform ID |
GPL16770 |
| Series (1) |
| GSE64629 |
MiRNAs expression profiles of nasopharyngeal carcinoma cell line HNE2 following TP53 overexpression |
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