特异性逆转录microRNA-505的高效茎环引物的设计

Design Specific and Efficient Reverse Transcription Stem-loop Primer of MicroRNA-505

目的:构建一个高效且特异性的逆转录茎环引物,以准确定量检测小鼠的mi RNA-505基因,同时探讨茎环引物的结构对逆转录效率的影响。方法:规律性地改变通用茎环引物的茎部和环部的碱基结构,再进行不同的组合,利用逆转录-聚合酶链式反应(Reverse Transcription Polymerase Chain Reaction,RT-PCR)技术,探寻效率最高的茎环引物。结果:当固定茎部结构为14对碱基,规律性改变环部碱基个数(15,18,21),经RT-PCR检测的CT值无差异,均为28.5;当固定环部结构为21个碱基,CT值随茎部碱基对(8,11,14)规律性地延长而逐渐增大;当环部结构为21个碱基,茎部结构为8对碱基时CT值最小,为26.7。结论:环部结构对茎环引物的效率无贡献;当环部结构为21个碱基,茎部结构为8对碱基时,茎环引物的效率和特异性是最佳的,适用于准确检测小家鼠的mi RNA-505基因。

Objective： A high efficient and specific stem-loop primer was designed to detect the mi RNA-505 gene accurately in mice and explore the effect of the stem-loop structure on the efficiency of reverse transcription. Methods： The structures of stem part and ring part of the general stem-loop primer were changed regularly and put in different combinations to select the optimum stem-loop primer based on RT-PCR（Reverse Transcription PCR） technology. Results： The results of RT-PCR showed that stem-loop primers,which was fixed stem structure（14 pairs of bases） and in various loop length（15,18,21bp）had the same CT value（28.5）;When the loop structure was 21 bp, the CT values were gradually increased while the length of stem extended（8,11,14 pairs of bases）; The minimum CT value was obtained when the stem part was 8 pairs of bases and the ring part was 21 bp. Conclusions： The loop structure has no effect on the reverse transcription. The primer which has 8 pairs of bases stem and 21 bp loop has the highest efficiency to the reverse transcription of mi RNA-505 gene in mice.