摘要
目的探索正交设计在实时定量PCR检测条件优化中的应用,找出扩增ICAM-1基因的最佳条件。方法建立大鼠皮肤损伤模型,采用正交设计实时定量PCR的各影响条件,提取损伤处皮肤组织总RNA反转录成cDNA,cDNA作为模板,通过特异性引物扩增ICAM-1基因。对扩增曲线和融解曲线制定评分标准,量化不同实验条件下的扩增结果并进行统计分析。结果退火温度、模板量和引物浓度直接影响扩增效果,但三因素间无相互作用。结果经统计学分析表明退火温度61℃、模板cDNA用量0.4μg和引物终浓度150μmol/L为实时定量PCR检测ICAM-1基因的最佳条件组合。结论正交设计是一种切实可行、快速经济的实时定量PCR检测条件优化方法。
Objective To investigate the application of orthogonal design to optimtze real-time quantita- tive PCR conditions and perform it for ICAM-1. Methods After model of a skin injury was established in rats, total RNA was isolated from the injured skin tissue and reverse transcriptase reactions were carried out. Using orthogonal design real-time quantitative PCR conditions,the expression level of ICAM-1 gene was studied by use of cDNA as template. The standard of score was set up to amplification and dissociation curves and statistical analysis was done based on the optimized conditions. Results Through 25 tests the best real-time quantitative PCR conditions for ICAM-1 are anneal temperature at 61℃, 0.41μg of cDNA and concentration of 150μmol/L each primer in the final reaction. There are no interaction among above factors. Conclusion Orthogonal design is a feasible, quick and economical method to optimize real-time quantitative PCR conditions.
出处
《中国法医学杂志》
CSCD
2008年第2期111-114,共4页
Chinese Journal of Forensic Medicine
基金
山西省留学回国人员基金资助项目(200528)
山西省青年科技研究基金项目(2007021047)
关键词
法医病理学
正交设计
实时定量PCR
皮肤损伤
ICAM-1
Forensic pathology
Orthogonal design
Real-time Quantitative Polymerase Chain Reaction
Skin injury
ICAM-1
