|
| Status |
Public on Jul 05, 2010 |
| Title |
C2C12_siRNA_Six4_R2 |
| Sample type |
RNA |
| |
|
| Source name |
C2C12, siRNA Six4
|
| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12
cell type: myoblasts
sirna treatment: siSix4
|
| Treatment protocol |
The cells were transfected, 24h before the start of differentiation, with 100 pmol of siRNA (Dharmacon) using the Lipofectamine2000 (Invitrogen) using the manufacturer's protocol.
|
| Growth protocol |
Cells were grown in growth medium (GM) DMEM High Glucose (Hyclone); 10% Characterized Fetal Bovine Serum (Hyclone); 1% L-Glutamine (Hyclone); 1% Penicillin / Streptomycin (Hyclone). At the start of differentiation, the cells were grown in differentiation medium (DM) DMEM High Glucose (Hyclone); 2% Horse Serum (Invitrogen); 1% L-Glutamine (Hyclone); 1% Penicillin / Streptomycin (Hyclone).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with the Absolutely RNA Mini-Prep Kit (Stratagene) following the manufacturer's protocol. RNA quantity and quality was assessed with the NanoDrop ND-1000. Integrity of the RNA was determined by agarose gel.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 300ng RNA using the Quick Amp Labeling Kit, one-color 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 |
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 55 ul containing 1x Agilent fragmentation buffer and 25x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and 100 ul of the mixture was hybridized to Agilent Whole Mouse Genome 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 air dried.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565C) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, 0.10 XDR, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene Expression profiling of C2C12 24h after start of differentiation when cells were transfected with an siRNA sequence against Six4.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1-v5_10_Apr08 and Grid: 014868_D_F_20080627) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Raw data were processed using GeneSpring GX (Agilent). Using the gMedian signal transformed to log base 2, the probes were normalized within the sample to the 75th percentile and the samples were then normalized by baseline transformation through the median of each sample.
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| |
|
| Submission date |
Jan 20, 2010 |
| Last update date |
May 10, 2023 |
| Contact name |
Alexandre Blais |
| E-mail(s) |
alexandre.blais@uottawa.ca
|
| Organization name |
University of Ottawa
|
| Street address |
451 Smyth Road
|
| City |
Ottawa |
| State/province |
ON |
| ZIP/Postal code |
K1H 8M5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE19967 |
Gene Expression Profiling of siRNA Knock-Down of Six1, Six4 and Myogenin in C2C12 |
| GSE19988 |
Six1 and Six4 function in myoblast differentiation |
|
