生物技术通报 ›› 2022, Vol. 38 ›› Issue (5): 279-285.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0746

• 技术与方法 • 上一篇    下一篇

基于跨反向剪接位点引物特异性检测circRNA的PCR方法

孙宝箴1(), 全龙萍1, 康慧1, 姚玉新1, 沈甜2, 陈卫平2, 杜远鹏1, 高振1()   

  1. 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
  • 收稿日期:2021-06-08 出版日期:2022-05-26 发布日期:2022-06-10
  • 作者简介:孙宝箴,男,硕士研究生,研究方向:葡萄抗逆栽培;E-mail: q864070339@163.com
  • 基金资助:
    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)

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

SUN Bao-zhen1(), QUAN Long-ping1, KANG Hui1, YAO Yu-xin1, SHEN Tian2, CHEN Wei-ping2, DU Yuan-peng1, GAO Zhen1()   

  1. 1. 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
    2. Institute of Horticulture,Ningxia Academy of Agriculture and Forestry Sciences,Yinchuan 750002
  • Received:2021-06-08 Published:2022-05-26 Online:2022-06-10

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。

关键词: circRNA, qRT-PCR, 背向引物, 可变反向剪接

Abstract:

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.

Key words: circRNA, qRT-PCR, divergent primer, alternative back-splicing