8 protocols
AccessionType
bioassay_data_transformation
Data were log2 transformed.
bioassay_data_transformation
We used loessM normalization. For the loessM normalization we adapted the method of Risso et al. (2009). In order to avoid small values close to 0 the median of the respondent value in the loess estimation is added to the dataset. This modification relaxes the assumption of symmetry among up- and down-regulated genes. We adopted the method for our one dimensional arrays. A brief description and the corresponding R code can be found in http://www.statistik.lmu.de/~kaiser/sup-material.html, respectively. Normalizations were done in R using the function: normalize.loessM (own code, http://www.statistik.lmu.de/~kaiser/sup-material.html).
image_acquisition
miRNA microarrays were scanned with the Agilent Microarray Scanner G2505C in a single pass mode with a scan resolution of 3 um, 20 bit mode. Signal intensities were extracted and background subtracted using Feature Extraction Software 10.7.3.1 (Agilent Technologies).
hybridization
Hybridization of labeled miRNAs were carried out by using the miRNA Complete Labeling and Hybridization Kit (Agilent Technologies, Cat. No. 5190-0456) and Mouse miRNA Microarrays Release 15.0, 8x15K (Agilent, G4471A-029152) according to the manufacturer's instructions.
labeling
Labeling (Cy3) of miRNAs were carried out by using the miRNA Complete Labeling and Hybridization Kit (Agilent Technologies, Cat. No. 5190-0456) according to the manufacturer's instructions.
grow
Mouse skeletal muscle cells (pmi28) were propagated in Hams F10 (PAA Laboratories), supplemented with 20 % FCS (Sigma-Aldrich), 2 mM L-glutamine (PAA Laboratories), and 1 % Penicillin / Streptomycin (PAA Laboratories). Myoblasts were propagated at 37 C in humidified air (80 % relative humidity) and 5 % CO2. Cells were cultured on laminin-1 coated dishes for an additional 24 h before switching a fraction of dishes to differentiation medium.
nucleic_acid_extraction
Total RNA was extracted with Trizol (Invitrogen) following manufacturer's instructions.
specified_biomaterial_action
Cells were cultured on laminin-1 coated dishes for an additional 24 h before switching a fraction of dishes to differentiation medium (DMEM medium containing 2 % horse serum (Gibco), 2 mM L-glutamine (PAA Laboratories), and 0.1 % gentamicin (Gibco)) with 2 x 103 U/ml mouse recombinant TNF-? (Roche Applied Science, Basel, Switzerland), or 5 ug/ml murine recombinant IGF-I, respectively. All media were replenished twice a day. pmi28 cells were harvested 24 h (myoblasts day one and myotubes day one without and with TNF-? or IGF-I, respectively,) after the induction of fusion by serum withdrawal.