Biology Reference
In-Depth Information
29.2.3
The mRNA Expression by Quantitative
Real-Time PCR Technique
The mRNA expression level of calcineurin inhibitory protein myocyte-enriched
calcineurin-interacting protein (MCIP) 1 in the differentiated myotube treated with
taurine and nifedipine was quantified by real-time PCR. Total RNA was extracted
from the harvested myotube using an RNeasy Plus Mini Kit (QIAGEN). Reverse
transcription was performed on 500 ng of total RNA using a PrimeScript ® RT
reagent Kit (TAKARA Bio. Inc. Shiga, Japan). Real-time quantitative PCR was
performed on cDNA aliquots with the FastStart DNA Master SYBR Green I and a
LightCycler (Roche Diagnostics, Mannheim, Germany). The sequences of the oli-
gonucleotide primer pairs used to amplify mRNA were as follows: MCIP1 forward
primer, 5¢ -CTTCAGAACATATGACAAGGAC-3 ¢; MCIP1 reverse primer,
5¢ -AGGTGTGAACTTCCTATGTGTA-3 ¢ ; b-actin forward primer, 5¢ -CCTGTA
TGCCTCTGGTCGTA-3¢; and b-actin reverse primer, 5¢ -AGACTTCGAGCA
GGAGATGG-3¢. A standard curve for each run was constructed by plotting the
crossover point against the log concentration. The concentration of target molecules
in each sample was then calculated automatically by reference to this curve
( r = −1.00), and results were standardized to the expression of b -actin. The speci fi city
of each PCR product was assessed by melting curve analysis.
29.2.4
Statistical Analysis
Statistical significances were determined by unpaired Student's t -test or one-way
ANOVA multiple comparison test. Data were expressed as the mean ± SD or the
median and plots of individual value. Differences were considered statistically
significant when the calculated P value was less than 0.05.
29.3
Results
29.3.1
The Effect of Taurine Treatment on the Differentiation
of C2C12 Myoblast to Myotube
The effect of taurine treatment on the differentiation of myoblast to myotube in
C2C12 cells was evaluated by the measurements of maximal length of short diameter
of differentiated myotube, the number of nucleus in the myotube, and the calculation
of fusion index which is the number of nucleus per the apsis length of examined
myotube. The maximal short diameter in the myotube was increased by taurine treat-
ments compared to that in the untreated control and was significantly higher in
20 mM taurine treatment than the control and 5 mM taurine treatment (Fig. 29.1a ).
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