|
| Status |
Public on Apr 29, 2024 |
| Title |
293T_siGFP_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
293T, GFP siRNA, 72hr
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: cultured human embryonic kidney cells
sirna: siGFP
|
| Treatment protocol |
In the HOTAIR experiment, siRNA oligonucleotides were targeted to HOTAIR or to GFP as a control. They were synthesized by Dharmacon. All transfections were performed by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Three independent knockdown experiments were performed with HOTAIR. Total RNA was extracted after 3 days to perform microarray analysis.
|
| Growth protocol |
HEK293T cells were cultured in DMEM (high glucose, Gibco) supplemented with 10% fetal bovine serum (Gibco), 10 mM NaHCO3 (Sigma) and penicillin/streptomycin (Gibco).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by a commercial kit (NucleoSpin, Macherey-Nagel®). The concentration of RNA was quantified by a Nanodrop 2000 spectrophotometer (Thermo) and the quality was monitored by the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Alexa fluor® 555
|
| Label protocol |
Using the Invitrogen SuperScript plus Indirect cDNA labeling System, we labeled 15ug total RNA with Alexa fluor® 555 and purified it using the purification module in the kit. Dye incorporation and cDNA yield were checked with the NanoDrop ND-1000.
|
| |
|
| Hybridization protocol |
Total Alexa fluor® 555 labeled cDNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the Agilent SurePrint G3 Human GE 8x60K array 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3µm, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
293T_siGFP_rep1
Gene expression after 72hr transfection, ctrl for siHOTAIR_rep1.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 028004_D_F_20100430) to obtain background subtracted and spatially detrended Processed Signal intensities. These data were processed in Agilent GX 11.5 . Detailed information is as follows:
Threshold: 1.0
Log base: 2
Technology: Agilent.SingleColor.28004
Normalization: Shift to 75 percentile
|
| |
|
| Submission date |
Feb 13, 2012 |
| Last update date |
Apr 29, 2024 |
| Contact name |
Hsiu-Ming Shih |
| E-mail(s) |
hmshih@ibms.sinica.edu.tw
|
| Organization name |
Academia Sinica
|
| Department |
Institute of Biomedical Sciences
|
| Street address |
128 Sec. 2, Academia Rd. Nankang,
|
| City |
Taipei |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE35792 |
Gene expression profiles of depletion of Daxx and HOTAIR |
|
