张驰宇1 ,2) , 张高红1 ,2) , 杨 敏1) , 贲昆龙1) 3
(1) 中国科学院昆明动物研究所分子与细胞免疫学实验室,昆明 650223 ;2) 中国科学院研究生院,北京 100039)
摘要 为建立一种新的SYBR green Ⅰ实时定量RT-PCR 方法,使之能够有效消除引物二聚体(PDs)对实时定量结果的影响. 对RT-PCR 特异性扩增产物和PDs 分别进行了凝胶电泳检测和熔解曲线分析. 依据PDs 和扩增产物的熔解温度( Tm) 特点,在通用的三步法的延伸步骤之后,增加一个短暂的(5 s) 恒温和荧光检测步骤,使这个步骤的温度高于PDs 的Tm ,但低于扩增产物的Tm ,简称该法为四步法. PDs 的Tm 通常高于72 ℃,但低于扩增产物的Tm . 将四步法第四步的温度设置在高于PDs 的Tm ,但低于扩增产物的Tm 时,四步法能够有效地消除PDs 的影响. 对三步法和四步法SYBR green Ⅰ实时定量RT-PCR 进行了比较,发现三步法根本不能用于RNA 的实时定量,而四步法能够实现包括低丰度RNA 在内的RNA 的定量. 选择Tm 值尽可能小的引物,使PDs 与扩增产物Tm 值之间有足够的差距,将更有利于四步法的应用,并可成功地用于低丰度RNA 的准确定量.
关键词 引物二聚体,荧光实时定量RT-PCR ,SYBR green Ⅰ,四步法,RNA 定量
Elimination of Primer2dimer Effect in SYBR Green Ⅰ Real-time
RT-PCR Using 42step Program
ZHANG Chi2yu1) , 2) , ZHANG Gao2hong1 , 2) , YANGMin1) , BEN Kun2long1) 3
(1) Laboratory for Molecular and Cell Immunology , Kunming Institute of Zoology , The Chinese Academy of Sciences , Kunming 650223 , China ;
2) The Graduate School of Chinese Academy of Sciences , Beijing 100039 , China)
Abstract To develop a new method for effectively eliminating the effect of primer2dimers ( PDs) in SYBR green Ⅰ real2time RT-PCR the specific amplicons and PDs formed by RT-PCR were measured by gel
electrophoresis with ethidium bromide staining ,and the characteristic of their melting temperatures( Tm) were analysed by melting curves. The so2called 42step program in SYBR green Ⅰreal2time RT-PCR was developed by adding a 5 s holding at the optimal temperature between Tm of PDs and amplicons for reading fluorescence signal after extension step of RT-PCR , and was compared with 32step programs on quantification of low abundance RNA. The 42step program effectively eliminated the artifact of PDs in SYBR green Ⅰreal2time RT-PCR. In addition , the 42step program may be used for quantifying low abundance RNA ,while the 32step program may not . The primer pair with low Tm should be preferably selected for the efficient elimination of the effect of PDs on quantification of low abundance RNA.
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