基于跨反向剪接位点引物特异性检测circRNA的PCR方法

doi:10.13560/j.cnki.biotech.bull.1985.2021-0746

摘要/Abstract

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

孙宝箴, 全龙萍, 康慧, 姚玉新, 沈甜, 陈卫平, 杜远鹏, 高振. 基于跨反向剪接位点引物特异性检测circRNA的PCR方法[J]. 生物技术通报, 2022, 38(5): 279-285.

SUN Bao-zhen, QUAN Long-ping, KANG Hui, YAO Yu-xin, SHEN Tian, CHEN Wei-ping, DU Yuan-peng, GAO Zhen. Back-splicing Primers-based PCR Method for Specific Detection of circRNA[J]. Biotechnology Bulletin, 2022, 38(5): 279-285.

图/表 10

表1 RT-PCR与荧光定量PCR所用引物

Table 1 Primers for RT-PCR and qRT-PCR

circRNA名称 Name of circRNA	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
circRNA_4363	AGAGATGCAGAACAACAGAGTTATG	GCGGGATTGAATCTGTGAATC
circRNA_6017	CAGAAGGATACCGTGGAATCGAT	ACTCTGCTGCAGCCTGTAGCAT
circRNA_6044	CATGCAACAGCTTTCAAGCAA	GAGCCTTCCTCCTTTCTTCCA
circRNA_7086	TTTGCTGTTTCTGGATACGCTTCT	TGCAGCATCAACTCTTTCATTGTC

表2 RT-PCR与荧光定量PCR改进引物

Table 2 Improved primers for RT-PCR and qRT-PCR

circRNA名称 Name of circRNA	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
circRNA_4363	ACTGATGGAGTTGATGAGGAA	GGTGTCCTTCTTCGGTTAAACT
circRNA_6017	GCTCTGTAAACTTAACCAGGAAA	GCATGCCATCTGAGATAATCCT
circRNA_6044	AAGAGAGAGCCAAGAAGGAGA	CCTTCTTGCTTGAAAGCTGT
circRNA_7086	CGTTTTTTCTCAGAGAAGAAGG	ATGATGGAAGGATCAGTACCGT

图1 circRNA可变反向剪接环化事件 a：并列关系;b：交叉关系;c、d：包含关系

Fig. 1 Alternative back-splicing circularization of circRNA a : Juxtaposition relationship. b : Crossover relationship. c and d : Inclusion relatio-nship

图2 VIT_201s0010g01480第7-13个外显子产生的circRNA

Fig. 2 circRNA produced by exons 7-13 of VIT_201s0010g01480

图3 circRNA_4363、circRNA_6017、circRNA_6044和circRNA_7086的RT-PCR结果

Fig. 3 RT-PCR results of circRNA_4363, circRNA_6017, circRNA_6044, and circRNA_7086 M: DL2000 marker

图4 circRNA反向剪接位点及其附近序列

Fig. 4 Back-splicing sites and nearby sequences of circRNA

图5 circRNA_4363、circRNA_6017、circRNA_6044和circRNA_7086的qRT-PCR溶解曲线

Fig. 5 qRT-PCR dissolution curve of circRNA_4363, circRNA_6017, circRNA_6044, and circRNA_7086

图6 改进后的引物设计方法

Fig. 6 Improved primer design method

图7 引物改进后circRNA_4363、circRNA_6017、circRNA_6044和circRNA_7086的RT-PCR结果

Fig. 7 RT-PCR results of circRNA_4363, circRNA_6017, circRNA_6044, and circRNA_7086 after using improved primers M: DL2000 marker

图8 引物改进后circRNA_4363、circRNA_6017、circRNA_6044和circRNA_7086的qRT-PCR溶解曲线

Fig. 8 qRT-PCR dissolution curve of circRNA_4363, circRNA_6017, circRNA_6044, and circRNA_7086 after using improved primers

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