|
| Status |
Public on Jul 01, 2021 |
| Title |
Cal33_siScr_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Cal33 transfected with siScr, repeat 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Cal33
treatment: siScr
|
| Treatment protocol |
Cells were seeded in 6-well plates at density 200000-300000 cells per well. Next day cells were transfected with 12ul Lipofectamine RNAiMax and 150 pmol of corresponding siRNA per well, and after 48h harvested for further analysis
|
| Growth protocol |
Standard tissue culture at 37 ºC in a humidified incubator with 5% CO2, in DMEM supplemented with 10% FBS, 10mM HEPES, and 1x Pen/Strep
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with RNeasy mini kit (Qiagen) following the manufacturer's recommendations
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer
|
| |
|
| 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 1x Agilent fragmentation buffer and 2x Agilent 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-039494 SurePrint G3 Human GE v2 8x60K Microarray 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 at 37°C wit 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 scan protocol Agilent G3_GX_1_color for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green)
|
| Description |
Gene expression in Cal33 transfected with siScr
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using protocol GE1_107_Sep09 and Grid: 072363_D_F_20150612 to obtain background subtracted and spatially detrended Processed Signal intensities
|
| |
|
| Submission date |
Apr 23, 2021 |
| Last update date |
Jul 01, 2021 |
| Contact name |
Vasyl Lukiyanchuk |
| E-mail(s) |
gmelyk@gmail.com
|
| Organization name |
OncoRay
|
| Street address |
Handelallee 26
|
| City |
Dresden |
| ZIP/Postal code |
01309 |
| Country |
Germany |
| |
|
| Platform ID |
GPL20844 |
| Series (1) |
| GSE173161 |
Comparative analysis of gene expression after Oct4 or Scrambled siRNA knockdown in HNSCC cell lines |
|
