|
| Status |
Public on Aug 06, 2021 |
| Title |
siCD271#2-2 |
| Sample type |
RNA |
| |
|
| Source name |
siCD271
|
| Organism |
Homo sapiens |
| Characteristics |
transfection: siCD271
cell type: human lung squamous cell carcinoma
cell line: MCC148c
|
| Treatment protocol |
The siRNA (siCD271 or si control) transfections were performed using Lipofectamine® RNAiMAX Reagent (Life Technologies, CA, USA) in antibiotic-free medium for 48 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from MCC148c cells was purified using RNeasy Mini Kits and QIAshredder columns (Qiagen, Canada).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 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, 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 (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction buffer 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 SurePrint G3 Human Gene Expression 8x60K(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), then dried immediately by Nitrogen gas.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 48hr lipofeation of sicontrol or siCD271 in lung sqmaous cell carcinoma cell line.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (GE1_107_Sep09) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Aug 06, 2018 |
| Last update date |
Aug 06, 2021 |
| Contact name |
Keiichi Tamai |
| E-mail(s) |
tamaikeiichi@med.tohoku.ac.jp
|
| Organization name |
Miyagi Cancer Center
|
| Street address |
1-47, Nodayama, Shiote, Medeshima
|
| City |
Natori, Miyagi |
| ZIP/Postal code |
981-1293 |
| Country |
Japan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE118141 |
CD271 is preferentially expressed and essential for cell proliferation in squamous cell carcinoma of the lung |
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