In a randomized, placebo-controlled, crossover, double-blind design, 12 young, healthy, non-obese men were supplemented with either a placebo or creatine monohydrate (loading phase, 20 g/d x 3 d; maintenance phase, 5 g/d x 7 d) for 10 d. Following a 28 day washout period, subjects were put on the alternate supplementation for 10 days. Muscle biopsies of the vastus lateralis were obtained and immediately placed in RNase-free cryoviles, flash-frozen in liquid nitrogen, and stored at -86°C until further analysis.
Extracted molecule
total RNA
Extraction protocol
The total RNA was extracted from the skeletal muscle biopsy as described previously in detail by our group (Mahoney et al., 2004). Briefly, ~25 mg of skeletal muscle was homogenized on ice in 1 mL of Trizol Reagent (Life Technologies, Cat. No.15596, Gaithersburg, MD). The homogenate was incubated for 10 min at room temperature, followed by phase separation using 200 μL of chloroform and precipitation of the total RNA from the aqueous phase using 500 μL of isopropyl alcohol. The RNA pellet was then washed three times in 75% ethanol and was re-suspended in 15 μL DEPC-treated water, aliquotted, and stored at -86°C. We assessed total RNA quality by measuring the size distribution on an Agilent Bioanalyzer (1.0-1.5 kb, Agilent Technologies, Inc., Santa Clara, CA) and by measuring the spectrophotometric 260/280 ratio (>1.8). Prior to cDNA microarray analysis, the isolated RNA samples were treated with DNA-free DNase I (Ambion Inc, Austin, TX) according to the manufacturer’s instructions to remove any potential genomic DNA contamination. mRNA was amplified in a linear fashion using MessageAmp aRNA kit (Ambion Inc, Austin, TX) according to the manufacturer’s instructions.
Label
Cy5
Label protocol
200 nanograms of amplified RNA was labeled using Genisphere's 350RP kit, which uses a two-step labeling process. First the aRNA is reverse transcribed and then ligated with a Cy3/Cy5 probe that has a capture sequence that will bind Cy3/Cy5.
Young Men wit Creatine Monohydrate Supplementation.
Treatment protocol
In a randomized, placebo-controlled, crossover, double-blind design, 12 young, healthy, non-obese men were supplemented with either a placebo or creatine monohydrate (loading phase, 20 g/d x 3 d; maintenance phase, 5 g/d x 7 d) for 10 d. Following a 28 day washout period, subjects were put on the alternate supplementation for 10 days. Muscle biopsies of the vastus lateralis were obtained and immediately placed in RNase-free cryoviles, flash-frozen in liquid nitrogen, and stored at -86°C until further analysis.
Extracted molecule
total RNA
Extraction protocol
The total RNA was extracted from the skeletal muscle biopsy as described previously in detail by our group (Mahoney et al., 2004). Briefly, ~25 mg of skeletal muscle was homogenized on ice in 1 mL of Trizol Reagent (Life Technologies, Cat. No.15596, Gaithersburg, MD). The homogenate was incubated for 10 min at room temperature, followed by phase separation using 200 μL of chloroform and precipitation of the total RNA from the aqueous phase using 500 μL of isopropyl alcohol. The RNA pellet was then washed three times in 75% ethanol and was re-suspended in 15 μL DEPC-treated water, aliquotted, and stored at -86°C. We assessed total RNA quality by measuring the size distribution on an Agilent Bioanalyzer (1.0-1.5 kb, Agilent Technologies, Inc., Santa Clara, CA) and by measuring the spectrophotometric 260/280 ratio (>1.8). Prior to cDNA microarray analysis, the isolated RNA samples were treated with DNA-free DNase I (Ambion Inc, Austin, TX) according to the manufacturer’s instructions to remove any potential genomic DNA contamination. mRNA was amplified in a linear fashion using MessageAmp aRNA kit (Ambion Inc, Austin, TX) according to the manufacturer’s instructions.
Label
Cy3
Label protocol
200 nanograms of amplified RNA was labeled using Genisphere's 350RP kit, which uses a two-step labeling process. First the aRNA is reverse transcribed and then ligated with a Cy3/Cy5 probe that has a capture sequence that will bind Cy3/Cy5.
Hybridization protocol
Using a Lucidea Slide Pro machine, a human cDNA array (custom print) is loaded into the slide chamber. The cDNA probe (Cy3 and Cy5 and/or Control and Experimental) is mixed with a formamide hybe buffer, dextran sulfate and LNA and Cot-1 DNA blocker and then loaded into the slide chamber and with a slow mixing program, the cDNA probe is circulated over the array for 18 hours at a temperature of 50degreesC. The array is then washed with 2XSSC+0.2%SDS, 2XSSC and then 0.2XSSC, afterwards, the dendromer (Cy3/Cy5) is mixed with hybe buffer, Cot DNA, dextran sulfate and anti-fade and loaded into the slide chamber. The fluorescent dendromer is captured by the bound cDNA sequences on the array. The probe is circulated for 3 hours at 50degreesC, and then washed and scanned.
Scan protocol
The array was scanned using a Scan Array Express scanner. Control spots from each channel were first normalized with a test scan, by adjusting the PMT and POW settings. Then the array was scanned at 10um. The fist channel was Cy5 and the second channel was Cy3.
Description
S15 - CrM vs. PL
Data processing
The Cy5 and Cy3 images are analyzed using GenePix software and a gal file.
