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Sample GSM499002 Query DataSets for GSM499002
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 (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.
Submission date Jan 20, 2010
Last update date May 10, 2023
Contact name Alexandre Blais
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

Data table header descriptions
VALUE normalized log2 signal

Data table
GE_BrightCorner -0.358572
DarkCorner -0.79936886
A_52_P616356 -1.0793772
A_52_P580582 -0.2513337
A_52_P403405 -0.83045197
A_52_P819156 -0.4872694
A_51_P331831 -0.52415514
A_51_P430630 -0.7477598
A_52_P502357 -0.86334515
A_52_P299964 -0.090883255
A_51_P356389 -0.72069407
A_52_P684402 0.1073885
A_51_P414208 -0.6456213
A_51_P280918 0.2694807
A_52_P613688 -0.9588957
A_52_P258194 -0.5291109
A_52_P229271 -1.1067817
A_52_P214630 0.21872711
A_52_P579519 -0.27684522
A_52_P979997 -1.0451183

Total number of rows: 41267

Table truncated, full table size 986 Kbytes.

Supplementary file Size Download File type/resource
GSM499002.txt.gz 2.2 Mb (ftp)(http) TXT
Processed data included within Sample table

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