|
| Status |
Public on Sep 01, 2012 |
| Title |
Proliferation before G0, Sample B |
| Sample type |
RNA |
| |
|
| Source name |
Sample B, Proliferating skeletal muscle myoblasts before G0 arrest
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Male
tissue: muscle biopsy obtained from m. Vastus Lateralis
cell line: myoblast cell culture B
culture condition: BG0
|
| Growth protocol |
Human muscle biopsies free of connective tissue were minced washed and dissociated with 0.05% trypsin-EDTA (Invitrogen) for 3x30 min. Harvested cells were pooled and Fetal Bovine Serum (FBS, Invitrogen) added as protease inhibitor. The isolated cells were seeded for up scaling (max 7 passages) on ECM-coated dishes (NUNC) after 15 min. of preplating and cultured in growth medium, GM (DMEM w. 10% FBS and 1% penicillin and streptomycin (PS), Invitrogen). Proliferating cells were G0 arrested in suspension medium (with 2% methyl cellulose (Sigma-Aldrich), 10% FBS and 1% PS), followed by reactivation in GM and finally differentiation in DM (DMEM with 2% FBS, 1% PS and 25 pmol Insulin (Actrapid from Novo Nordisk)) for 5-7 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultured cells were rinsed twice in PBS and lysed in 1x Nucleic Acid Purification Lysis Solution (Applied biosystems, Foster City, CA, USA). Samples from suspension cultures were collected at various time points, washed twice in PBS and lysed with Lysis Solution. RNA was isolated using ABI PRISMTM 6100 Nucleic Acid PrepStation with Total RNA Chemistry kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
The samples for Gene expression were labeled using Agilent Low RNA Input Linear amplification Kit (Part number:5184-3523). Five hundred nanograms each of the Control and test samples were incubated with reverse trancription mix at 40°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields and specific activity.
|
| |
|
| Hybridization protocol |
1650ng of cy3 labeled samples were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). Hybridization was carried out in Agilent's Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327)
|
| Scan protocol |
Scanned on an Agilent G2505B scanner and Images were quantified using Agilent Feature Extraction Software (version 9.5.3.1)
|
| Description |
Gene expression in myoblasts proliferating before G0 arrest
Sample name: FC [BG1]
|
| Data processing |
The normalization was done using GeneSpring GX 10 Software. Recommended Percentile Shift Normalization.
|
| |
|
| Submission date |
Jun 18, 2012 |
| Last update date |
Sep 01, 2012 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE38769 |
Gene expression in primary isolated human myoblast during proliferation, G0 arrest, reactivation and differentiation |
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