The si RNA sequences were designed using Invitrogen’s BLOCK-iT RNAi designer. The primer sequences targeting MGP corresponding to the coding region 348 to 366 were 5’-GGACUUCCACCUGUGUGAA-3’ (sense) and UUCACACAGGUGGAAGUCC-3’ (antisense). The siRNAs were purchased from Invitrogen in the desalted, pre-annealed duplex form. The transfection of the siRNAs into the satellite cells were done using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommended conditions.
Growth protocol
Satellite cells were isolated from the pectoralis major muscle of 7-week-old male RBC2 line turkeys and plated in 24-well gelatin-coated cell culture plates in antibiotic free plating media. Satellite cell cultures were transfected cells with the appropriate siRNA or mock-transfected to a final concentration of 20 pM of each siRNA and 1.5 µL lipofectamine 2000 (Invitrogen) contained in 100 µL Opti-MEM I Reduced Serum medium (Invitrogen) per well. The transfection mixture was added to the cells and incubated for 5 hours at 37 °C in a 95% air/5% CO2 incubator. After incubation, the medium was replaced with growth medium containing McCoy’s 5A (Sigma-Aldrich, St. Louis, MO) containing 10% chicken serum (Gemini BioProducts, West Sacramento, CA), and 5% horse serum (Gemini BioProducts) with 0.1% antibiotic/antimycotic (Gemini BioProducts). The growth medium was changed every 24 hours until the cells reached 60-65% confluency. Differentiation was induced by changing the medium to Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) containing 3% horse serum, 0.1 mg/mL porcine gelatin (Sigma-Aldrich), and 1.0 mg/mL BSA (Sigma-Aldrich) with 10 µg/mL gentamicin (Invitrogen), and 0.1% antibiotic/antimycotic. The differentiation medium was changed daily. After 48 h of differentiation, the plates were removed, washed with PBS, and stored at -70 °C until analysis.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cell culture plates (n = 4) using TRIReagent (Molecular Research Center, Cincinnati, OH) following manufacturer's instructions. RNA integrity and concentration was determined using an Agilent 2100 Bioanlyzer (Santa Clara, CA).
Label
Cy3
Label protocol
Total RNA for use in microarrays was amplified using two rounds of amplification with the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion, Inc.) per manufacturer instructions. Briefly, 1 μg total RNA was incubated with T7 Oligo(dT) primer for 10 min at 70°C then reverse transcribed into cDNA at 42°C for 2 h. Second strand synthesis was performed at 16°C for 2 h. Double-stranded cDNA was then purified, and in vitro transcription was performed at 37°C for 6 h. The resulting aRNA was purified and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). For the dye coupling procedure, 15 μg aRNA was coupled to either Cy3 or Cy5 fluorescent dye (GE Healthcare, Piscataway, NJ) in the dark at room temperature for 1 hour until the reaction was quenched by the addition of 4M hydroxylamine. Dye-coupled aRNA was purified and quantified, then 5 μg was fragmented to 60-200 nucleotide segments using RNA Fragmentation Reagents (Ambion, Inc.) at 70°C for 15 min in preparation for microarray hybridization.
The si RNA sequences were designed using Invitrogen’s BLOCK-iT RNAi designer. The primer sequences targeting MGP corresponding to the coding region 348 to 366 were 5’-GGACUUCCACCUGUGUGAA-3’ (sense) and UUCACACAGGUGGAAGUCC-3’ (antisense). The siRNAs were purchased from Invitrogen in the desalted, pre-annealed duplex form. The transfection of the siRNAs into the satellite cells were done using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s recommended conditions.
Growth protocol
Satellite cells were isolated from the pectoralis major muscle of 7-week-old male RBC2 line turkeys and plated in 24-well gelatin-coated cell culture plates in antibiotic free plating media. Satellite cell cultures were transfected cells with the appropriate siRNA or mock-transfected to a final concentration of 20 pM of each siRNA and 1.5 µL lipofectamine 2000 (Invitrogen) contained in 100 µL Opti-MEM I Reduced Serum medium (Invitrogen) per well. The transfection mixture was added to the cells and incubated for 5 hours at 37 °C in a 95% air/5% CO2 incubator. After incubation, the medium was replaced with growth medium containing McCoy’s 5A (Sigma-Aldrich, St. Louis, MO) containing 10% chicken serum (Gemini BioProducts, West Sacramento, CA), and 5% horse serum (Gemini BioProducts) with 0.1% antibiotic/antimycotic (Gemini BioProducts). The growth medium was changed every 24 hours until the cells reached 60-65% confluency. Differentiation was induced by changing the medium to Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) containing 3% horse serum, 0.1 mg/mL porcine gelatin (Sigma-Aldrich), and 1.0 mg/mL BSA (Sigma-Aldrich) with 10 µg/mL gentamicin (Invitrogen), and 0.1% antibiotic/antimycotic. The differentiation medium was changed daily. After 48 h of differentiation, the plates were removed, washed with PBS, and stored at -70 °C until analysis.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cell culture plates (n = 4) using TRIReagent (Molecular Research Center, Cincinnati, OH) following manufacturer's instructions. RNA integrity and concentration was determined using an Agilent 2100 Bioanlyzer (Santa Clara, CA).
Label
Cy5
Label protocol
Total RNA for use in microarrays was amplified using two rounds of amplification with the Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion, Inc.) per manufacturer instructions. Briefly, 1 μg total RNA was incubated with T7 Oligo(dT) primer for 10 min at 70°C then reverse transcribed into cDNA at 42°C for 2 h. Second strand synthesis was performed at 16°C for 2 h. Double-stranded cDNA was then purified, and in vitro transcription was performed at 37°C for 6 h. The resulting aRNA was purified and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). For the dye coupling procedure, 15 μg aRNA was coupled to either Cy3 or Cy5 fluorescent dye (GE Healthcare, Piscataway, NJ) in the dark at room temperature for 1 hour until the reaction was quenched by the addition of 4M hydroxylamine. Dye-coupled aRNA was purified and quantified, then 5 μg was fragmented to 60-200 nucleotide segments using RNA Fragmentation Reagents (Ambion, Inc.) at 70°C for 15 min in preparation for microarray hybridization.
Hybridization protocol
Fragmented, Cy3-coupled aRNA was mixed with its Cy5-coupled partner, and the mixtures were brought to 110 μL with 68°C SlideHyb 1 (Ambion, Inc.) and incubated at 68°C for 5 min. These dye-coupled mixtures were then hybridized to oligonucleotide microarrays (described previously) for 18 h in a GeneTac Hybridization Station (Genomic Solutions, Ann Arbor, MI) using the following conditions: 54°C for 18 h, followed by a medium-stringency wash at 42°C (2X SSC, 0.1% SDS), a high-stringency wash (0.2X SSC, 0.1% SDS) at 22°C, and a wash with postwash buffer (0.2X SSC) at 22°C. Arrays were then rinsed in 2X SSC and Nanopure H2O, dried by centrifugation at 500 × g for 5 min.
Scan protocol
Scanned with a GenePix 4000B (Molecular Devices, Sunnyvale, CA) scanner. Image analysis was performed using GenePix Pro 6.0, and spot intensities were exported as GPR files.
Description
MGP Knockdown versus Control, 48h differentiation, cell culture plate 2, replicate 3 of 8.
Data processing
Fluorescence intensity data was normalized for dye intensity bias using the LOESS procedure of the normalizeWithinArray function of the Bioconductor R software LIMMA (Smyth 2004).
