Animal Experimentation
The present experiment was conducted with mice that were MSTN-/- [13], MSTN+/+ and MSTN+/- (either sex). MSTN+/- mice were mated to produce MSTN-/- (n = 9), MSTN+/+ (n = 10) and MSTN+/- (n = 10) animals. The mice were housed in a temperature- and humidity-controlled room and maintained at 22°C on a 12-h light-dark cycle with food and water provided ad libitum. At 1 week of age, mice were individually identified and genotyped. Mice were weaned at 21 days of age. At 35 days of age mice were euthanized by CO2 asphyxiation. The pectoralis muscles were excised, flash-frozen in liquid nitrogen, and stored at -80°C until RNA isolation. The Iowa State University Institutional Animal Care and Use Committee approved all procedures that involved mice.
DNA Isolation and Genotyping
The DNA was isolated by standard protocol with few modifications[14] from toes clipped at seven days after birth. Myostatin genotyping was completed with a three-primer PCR reaction. The primer sequences were: Mstn1-5'-GGC ATC TGT TCT GCT ATT ACG TGC-3', Mstn2-5'-GTG CGA TAA TCC AGT CCC AT-3' and Mstn3-5'-GTG GAT GTG GAA TGT GTG CGA GG-3'. The PCR amplification reaction contained 50 ng DNA, 0.2 μl (50 pmol) of each primer, 5 μl of 2X Green GoTaq® Reaction Buffer (pH 8.5) (GoTaq® DNA Polymerase, 400 μM dATP, 400 μM dGTP, 400 μM dCTP, 400 μM dTTP and 3 mM MgCl2). The PCR amplification was carried out in a programmable thermal cycler (MJ research) using the following program: 3 minutes at 92°C, 15 seconds at 92°C, 30 seconds at 65°C, 30 seconds at 72°C followed by 39 cycles of 15 seconds at 92°C, 30 seconds at 65°C, 30 seconds at 72°C, final extension of 10 minutes at 72°C. The PCR products were size separated on 1.5% agarose gels to confirm genotype based on the amplification of target regions.
RNA Isolation
Total RNA was isolated from left pectoralis muscle using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's directions. The RNA was purified by columns (Qiagen sciences, Maryland, USA) and treated with DNase I (Qiagen, Maryland, USA) to remove genomic DNA contamination. The RNA concentration was measured by UV absorbance. RNA samples with an A260/280 ratio of = > 2.0 were used for RT-PCR.
cDNA Synthesis and Real Time PCR for MYOD and Myogenin
Reverse transcription of RNA was performed (Invitrogen, Carlsbad, CA) by adding 10 μl of (500 ng) total RNA, 1 μl of Oligo (dT) 12-18 (500 μg/ml), and 1 μl 10 mM dNTP incubated at 65°C for 5 minutes and immediately chilled on ice. Then, each component was added in the indicated order 4 μl of (5×) first strand RT buffer, 0.1 M DTT 1 μl, RNaseOUT. RNase Inhibitor 1 μl, incubated at 42°C for 2 min. 1 μl (50 units) of SuperScript II RT was then added to each tube mix, and incubated at 42°C for 50 min. The reactions were terminated at 70°C for 15 min. Chill on ice.
Real-time PCR was performed on Stratagene 4000× Real-Time PCR System. Quantitative PCR reactions were optimized for annealing temperature and primer concentration. Real-time PCR reactions were performed in triplicate and template controls (NTCs) were run for each assay under the same conditions. End-point PCR was then performed in 10 μl reaction containing 5 μl SBYR green Master Mix, 3.2 μl of autoclaved nuclease free water, 1 μl diluted RT product (Minimum1:10 but MyOD undiluted cDNA was used) and 0.4 μl forward and reverse primers (specific to genes i.e MyoD F-5'-TACAGTGGCGACTCAGATGC-3' and R-5'-GCTCCACTATGCTGGACAGG-3' and MyoG F-5'-CTACAGGCCTTGCTCAGCTC-3' and R-5'-ACGATGGACGTAAGGGAGTG-3') for 40 cycles (two steps: 95°C for 10 minutes followed by 40 cycles 95°C 15s followed by 58°C for 60 s). Standard curves were generated with known amounts (molecules) of cDNA and run with each PCR run. Gene expression levels were normalized to the level of β-actin expression, which we have shown to be unresponsive to Myostatin expression level [15], and are reported relative to Myostatin wild-type expression level.
Detection of Mature miRNAs by TaqMan assays
Five mouse Taqman miRNA assays were purchased from Applied Biosystems. Reverse transcription reactions (15 μl) containing, 1.5 μl of 10× reverse transcription buffer, 1.0 μl of MultiScribe™reverse transcriptase (50 U/μl), 0.15 μl of 100 mM dNTPs (with dTTP) and 0.19 μl of RNase inhibitor (20 U/μl), 4.16 of Nuclease-free water were mixed by brief centrifugation, 5 μl of total RNA (50 ng) was added and mixed well, 3 μl of 1× looped-primers were added (specific for microRNA) and mixed well and incubated for 30 min each, at 16°C, 42°C and 5 minutes at 85°C according to manufacturer's directions. The cDNA was diluted to a minimum of 1:15 with nuclease free water and 1.33 μl was used in real time PCR.
Real-time PCR was performed on Stratagene 4000× Real-Time PCR System. During the target amplification step, the AmpliTaq® Gold DNA polymerase amplifies target cDNA synthesized from the RNA sample, using sequence-specific primers from the TaqMan Assay. Real-time PCR reactions based on TaqMan® reagent chemistry were performed in triplicate and template controls were run for each assay under the same conditions. End-point PCR was then performed in 20 μl reactions that contained 10.00 μl TaqMan 2× Universal PCR Master Mix No AmpErase UNG, 7.67 μl of autoclaved nuclease free water, 1.33 μl diluted RT product (Minimum1:15) and 1 μl miRNA-specific PCR probe for 40 cycles (two steps: 95°C for 10 minutes followed by 40 cycles 95°C 15s followed by 60°C for 60 s). miRNA expression levels were normalized to the level of miR-24 expression, which was unresponsive to Myostatin expression level, to correct for differential cDNA content.
Statistical analysis
Student's t-test was used to determine whether a significant difference existed among MSTN-/-, MSTN+/- and MSTN+/+ animals. The P < 0.05 was denoted by an asterisk.
