双内参基因用于大鼠挫伤肌肉TAB2 mRNA表达量检测

Application of multiple reference genes for TAB2 mRNA expression in rat skeletal muscle

目的比较和分析Rpl32和Rpl13作为单内参基因与两者作为双内参基因,用于检测大鼠挫伤肌肉组织中TAB2 m RNA表达量的结果。方法 78只SD大鼠,随机分为正常对照组(1组)和肌肉损伤组(12组)。采用自由落体法制作大鼠肌肉挫伤模型,分别于4h^48h之间12个时间点取肌肉组织,提取总RNA、合成c DNA、采用real-time q PCR法,以Rpl32和Rpl13作为单独内参基因和二者作为双内参基因检测大鼠肌肉挫伤组织中TAB2 m RNA相对表达量,并计算检测结果的变异系数。结果以Rpl32或Rpl13单独作为内参基因或作为双内参基因,TAB2 m RNA表达随损伤时间总体均呈现降低的变化规律,但使用单内参基因在24h和32h出现极高值,而用双内参基因计算未出现极值,且各结果的变异系数均较小。结论 Rpl32和Rpl13作为双内参基因用于计算TAB2 m RNA相对表达量,可提高分析结果的准确性,在相关实验中建议选择使用。

Objective Comparison and analysis of the relative quantitative results using single reference gene or multiple reference genes for real-time quantification PCR method to detect TAB2 mRNA expression in rat contused skeletal muscle tissue. Methods A total of 78 SD rats were randomly divided into normal control group （lgroup） and muscle injury group （12 Groups）. Free-fall method was used to produce muscle contusion model of rat, the muscle tissue were taken at 12 time points between 4h-48h. Total RNA was extracted from the muscle tissue and cDNA was synthesized, Rp132 and Rpll3 were used both single reference gene and multiple reference genes for real-time qPCR method to detect TAB2 mRNA expression in rat contused muscle, and the variation coefficient of test results of each group were used to demonstrate the tendency of dispersion. Results TAB2 mRNA expression presented a decrease tendency after injury when using Rp132 or/and Rpll3 as the reference genes respectively, but the extreme values appeared at 24h and 32h after injury when used single reference gene. Otherwise, when multiple reference genes were used to normalize the data, the extreme values were eliminated and the coefficient of variation was smaller at most groups. Conclusion The result was more accurate when using multiple reference genes for calculating TAB2 mRNA relative quantity expression. And the result suggested that multiple reference genes should be widely applied to the same type research for estimating the wound age.