|
| Status |
Public on Mar 19, 2010 |
| Title |
CD44 siRNA, 1 |
| Sample type |
RNA |
| |
|
| Source name |
HCT116 cells, CD44 siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
cell type: colon cancer
sirna: CD44
|
| Treatment protocol |
Cells were transfected with the annealed siRNAs for 72 h with the use of the Lipofectamine RNAi MAX reagent (Invitrogen) according to manufacturer's recommendations.
|
| Growth protocol |
HCT116 cells were cultured under 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium–nutrient mixture F-12 supplemented with 10% fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the NucleoSpin RNA XS (MACHEREY-NAGEL GmbH & Co. KG,Düren) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
25ng total RNA was amplified by MessageAmp™ II aRNA Amplification Kit#1751(Ambion), and 0.2 ug aRNA labeled Cyanine-3 (Cy3) using the One-Color Quick Amp Labering 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.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55ul containing 25x fragmentation buffer and 10x blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55ul of 2xGE hybridization buffer(HI-RPM) was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Oligo Microarrays (G4122F) 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 (G2565BA) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR Hi 100% and XDR Lo 10% ).
|
| Description |
Gene expression data from HCT116 cells transfected with CD44 siRNA.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.5 (Agilent) using default parameters (protocol GE1-v5_10_Apr08 and Grid: 014868_D_F_20070820) 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 |
Mar 16, 2010 |
| Last update date |
Mar 18, 2010 |
| Contact name |
Osamu Nagano |
| E-mail(s) |
osmna@sb3.so-net.ne.jp
|
| Phone |
+81-3-5363-3983
|
| Fax |
+81-3-5363-3982
|
| Organization name |
Keio University School of Medicine
|
| Department |
Institute for Advanced Medical Research
|
| Lab |
Division of Gene Regulation
|
| Street address |
３５ Shinanomachi
|
| City |
Tokyo |
| State/province |
DDS PhD |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE20914 |
Expression of HCT116 cells transfected with CD44 siRNAs relative to that of HCT116 cells transfected with a control siRNA |
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