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Status |
Public on Nov 17, 2011 |
Title |
C2C12_cells_treated_with_rhBMP2 |
Sample type |
RNA |
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Source name |
C2C12 cells treated with rhBMP2
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Organism |
Mus musculus |
Characteristics |
cell type: pre-myoblastic and pre-osteoblastic C2C12 cell line
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Biomaterial provider |
American Type Culture Collection
|
Treatment protocol |
C2C12 cultures treated for 12h with 200ng/ml rhBMP2
|
Extracted molecule |
total RNA |
Extraction protocol |
total RNA was extracted with RNeasy The cRNA was prepared as recommended in the Amersham CodelinkTM iExpress Assay Reagent Kit manual. Briefly, the sample containing 1.5 µg RNA of culture treated for 12h with rhBMP2 was mixed with 7.5 µl of bacterial mRNAs (10pg/µl), 1 µl of oligo dT in a final volume of 12 µl. The reaction was incubated at 70ºC for 10min and kept on ice by 3min. Subsequently, 2 µl of 10X First Strand Buffer, 4 µl 5mM dNTPs, 1 µl RNase inhibitor, and 1 µl ArrayScript were added to tube, in a final volume of 20 µl. The reaction was incubated at 42°C for 2h. After this period, the tube was placed on ice and 63 µl water, 10 µl of 10X Second Strand Buffer, 4 µl of 5mM dNTPs, 2 µl of DNA polymerase and 1 µl of RNaseH were added. The reaction is incubating at 16°C for 2h.
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Label |
biotin
|
Label protocol |
The cDNA was purified and 20 µl of each purified cDNA was mixed with 12 µl Biotin- NTP, 4µl of 10X T7 reaction buffer and 4 µl of 10X Enzyme Mix reaction buffer and incubated at 37°C for 14h. After cRNA synthesis, the cRNA preparation was purified and 10 µg of biotinylated cRNA was mixed with 5 µl of Fragmentation Buffer in a final volume of 25 µl , incubated at 94°C for 20min and then kept on ice for 5min.
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Hybridization protocol |
25µl of reaction was transferred to another tube containing 78µl of Hybridization Buffer A, 130µl of Hybridization Buffer B and 27µl of water and the reaction was incubated at 90°C for 5min. Each tube was then placed on ice and then applied to the Codelink DNA microarray containing around 36,000 genes. The subsequent stages of hybridization, washing and analysis of each microarray were carried out as detailed in the Codelink Gene Expression System: Single-Assay Biorray Hybridization and Detection manual.
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Scan protocol |
Scanner Gene Pix 4000B (Molecular Devices) standard procedure
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Description |
n/a
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Data processing |
The gene expression date were normalized using the Suport Vector Regression (SVR). Implemented in the GEDI toolbox (http://www.iq.usp.br/wwwdocentes/mcsoga/gedi/) . Identification of these genes was obtained using the ratio of normalized intensities between rhBMP2 and control (untreated) samples, considering a fold change of >= 2.5.
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Submission date |
Aug 24, 2010 |
Last update date |
Nov 17, 2011 |
Contact name |
Maria Angélica Lopes de Souza |
Organization name |
Scylla Bioinformática
|
Street address |
Franscisco Otaviano nº 60 sl 52
|
City |
Campinas |
State/province |
São Paulo |
ZIP/Postal code |
13083765 |
Country |
Brazil |
|
|
Platform ID |
GPL8063 |
Series (1) |
GSE23783 |
Genes modulated by rhBMP2 and rhBMP7 during osteoblastic differentiation of C2C12 cells |
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