Article Summary
熊黎 仝莉 王茂春 周宇荀 李凯 肖君华.特异性逆转录microRNA-505 的高效茎环引物的设计[J].现代生物医学进展英文版,2015,15(27):5225-5229.
特异性逆转录microRNA-505 的高效茎环引物的设计
Design Specific and Efficient Reverse TranscriptionStem-loop Primer of MicroRNA-505
  
DOI:
中文关键词: 茎环引物  miR-505  逆转录  逆转录-聚合酶链式反应(RT-PCR)
英文关键词: Stem-loop primer  MiR-505  Reverse transcription  RT-PCR
基金项目:中央高校基本科研业务费专项资金(13D110521);国家自然科学基金项目(31171199)
Author NameAffiliation
熊黎 仝莉 王茂春 周宇荀 李凯 肖君华 东华大学生物科学与技术研究所 
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中文摘要:
      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miRNA-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miRNA-505 基因。
英文摘要:
      Objective:A high efficient and specific stem-loop primer was designed to detect the miRNA-505 gene accurately in mice and explore the effect of the stem-loop structure on the efficiency of reverse transcription.Methods:The structures of stempart 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 stemextended(8,11,14 pairs of bases); The minimumCT value was obtained when the stem part was 8 pairs of bases and the ring part was 21 bp.Conclusion:The loop structure has no effect on the reverse transcription. The primer which has 8 pairs of bases stem and 21bp loop has the highest efficiency to the reverse transcription of miRNA-505 gene in mice.
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