基于跨反向剪接位点引物特异性检测circRNA的PCR方法  

Back-splicing Primers-based PCR Method for Specific Detection of circRNA

作　　者：孙宝箴 全龙萍 康慧 姚玉新 沈甜[2] 陈卫平[2] 杜远鹏 高振 SUN Bao-zhen;QUAN Long-ping;KANG Hui;YAO Yu-xin;SHEN Tian;CHEN Wei-ping;DU Yuan-peng;GAO Zhen(College of Horticulture Science and Engineering,Shandong Agricultural University/Collaborative Innovation Center of Fruit&Vegetable Quality and Efficient Production in Shandong/State Key Laboratory of Crop Biology,Tai'an 271018;Institute of Horticulture,Ningxia Academy of Agriculture and Forestry Sciences,Yinchuan 750002)

机构地区：[1]山东农业大学园艺科学与工程学院山东果蔬优质高效生产协同创新中心作物生物学国家重点实验室,泰安272018 [2]宁夏农林科学院园艺研究所,银川750002

出　　处：《生物技术通报》2022年第5期279-285,共7页Biotechnology Bulletin

基　　金：山东省自然科学基金青年项目(ZR2020QC148);宁夏农业关键技术攻关项目;国家现代农业产业技术体系资助(CARS-29-zp-2)。

摘　　要：为了特异扩增circRNA发生可变反向剪接环化事件时被包含的circRNA,准确分析目标circRNA的表达水平。首先通过生物信息学分析葡萄高可信度circRNA的可变反向剪接环化事件特征,提出一种适用于葡萄circRNA可变反向剪接环化事件的引物设计改良方法,即一端引物的3'端跨反向剪接位点3-4个碱基,并运用RT-PCR和qRT-PCR方法进行验证。结果表明,高可信度葡萄circRNA的来源基因中有21.7%存在可变反向剪接环化事件,可分为并列、交叉和包含关系。选取4组circRNA进行扩增,其中circRNA_4363、circRNA_6017、circRNA_6044和circRNA_7086分别被circRNA_4364、circRNA_6018、circRNA_6045和circRNA_7085包含,使用常规背向引物对被包含的4个circRNA进行RT-PCR可获得2条扩增条带,且qRT-PCR溶解曲线为双峰。通过应用改良引物对circRNA_4363、circRNA_6017和circRNA_7086进行RT-PCR和qRT-PCR可获得单一扩增产物。相较于常规背向引物,使用改良引物可以特异扩增发生可变反向剪接环化事件时被包含的circRNA,使其定量分析结果更加准确可靠。In order to specifically amplify the contained circRNA in alternative back-splicing circularization events and accurately analyze the expression level of the target circRNA,we firstly analyzed the characteristics of the alternative back-splicing circularization events of high-confidence circRNA in grape(Vitis vinifera L.)through bioinformatics,and further proposed an improved method of primers design suitable for the alternative back-splicing circularization events,that is,the 3'end of one primer spanned 3-4 bases of the back-splicing junction.Moreover,we verified it via RT-PCR and qRT-PCR.The results showed alternative back-splicing circularization events were found in 21.7%of the source genes of high-confidence grape circRNA,which could be divided into juxtaposition,crossover and inclusion relationships.We selected 4 groups of circRNA for amplification,circRNA_4363,circRNA_6017,circRNA_6044 and circRNA_7086 were contained by circRNA_4364,circRNA_6018,circRNA_6045 and circRNA_7085,respectively.Two amplified bands were obtained by RT-PCR of the 4 contained circRNAs with conventional divergent primers,and the qRT-PCR dissolution curve was bimodal.A single amplified product can be obtained by using modified primers to perform RT-PCR and qRT-PCR on circRNA_4363,circRNA_6017 and circRNA_7086.Compared with conventional divergent primers,using modified primers specifically amplified the contained circRNA in alternative back-splicing circularization events,making the quantitative analysis results more accurate and reliable.

关 键 词：circRNA QRT-PCR 背向引物 可变反向剪接 

分 类 号：Q503[生物学—生物化学]