预扩增qPCR方法检测少量小鼠早期胚胎细胞中DNA甲基化相关基因的表达

doi:10.3969/j.issn.1004-616x.2016.01.011

癌变·畸变·突变 ›› 2016, Vol. 28 ›› Issue (1): 51-55.doi: 10.3969/j.issn.1004-616x.2016.01.011

预扩增qPCR方法检测少量小鼠早期胚胎细胞中DNA甲基化相关基因的表达

程琳^1,2,3, 孙平楠^1,3, 谢庆东^1,2, 周小玲^1,2,3

1. 汕头大学医学院干细胞P2实验室, 广东汕头 515041;
2. 汕头大学医学院生殖医学中心, 广东汕头 515041;
3. 广东省感染病与分子免疫病理重点实验室, 广东汕头 515041

收稿日期:2015-05-05 修回日期:2015-12-24 出版日期:2016-01-31 发布日期:2016-01-31
通讯作者: 周小玲,E-mail:xiaolingsmiling_zhou@yahoo.com E-mail:xiaolingsmiling_zhou@yahoo.com
作者简介:程琳,E-mail:470751814@qq.com。
基金资助:
国家自然科学基金(81570567,81571994);广东省自然科学基金(2014A030313483,2015A030313447);教育部留学回国人员科研启动基金(第49批);汕头大学医学院李嘉诚基金

Detection of expression of methylation-related genes in mouse early embryonic cells using pre-amplification-based real time quantitative PCR

CHENG Lin^1,2,3, SUN Pingnan^1,3, XIE Qingdong^1,2, ZHOU Xiaoling^1,2,3

1. Stem Cell P2 Laboratory, Shantou University Medical College, Shantou 515041;
2. Reproductive Medicine Center, Shantou University Medical College, Shantou 515041;
3. Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou 515041, Guangdong, China

Received:2015-05-05 Revised:2015-12-24 Online:2016-01-31 Published:2016-01-31

摘要/Abstract

摘要： 目的: 比较3种实时定量PCR(qPCR)方法对少量小鼠早期胚胎细胞中甲基化相关基因表达水平的检测效果,以期获得适合日常检测的方法。方法: 分别应用常规qPCR方法、基于等温预扩增的qPCR方法、基于PCR预扩增的qPCR方法3种方案对少量小鼠早期胚胎细胞样本中甲基化相关基因TET1、TET2、TET3、DNMT3A的mRNA表达进行检测。结果: 3种方法的灵敏度由高到低依次为基于等温预扩增的qPCR方法、基于PCR预扩增qPCR方法、常规qPCR方法。前两种方法均能在合适的循环数下对少量小鼠胚胎细胞中的多个甲基化相关基因表达进行定量。而基于PCR预扩增的qPCR方法比基于等温预扩增的qPCR方法更经济,适合日常检测。因此,我们选择应用基于PCR预扩增的qPCR方法,检测了小鼠卵细胞以及卵细胞受精后22 h甲基化相关基因表达的变化,结果发现该过程中TET1表达量很低,不易检测到;TET2表达呈降低趋势;TET3和DNMT3A表达显著升高(P<0.01),与文献报道基本一致。结论: 基于等温预扩增的qPCR方法检测少量细胞样品中甲基化相关基因的表达的灵敏度在3种qPCR方法中最高。而基于PCR预扩增的qPCR方法操作简单、灵敏度较高、成本相对低,适合少量细胞样本中多个相关基因表达的实时定量日常检测。

关键词: 预扩增, 实时定量PCR, 小鼠, 早期胚胎细胞, 甲基化相关基因

Abstract: OBJECTIVE: To identify a proper qPCR method for examining expression of methylation-related genes in mouse early embryonic cells. METHODSNormal qPCR,isothermal pre-amplification-based qPCR,and PCR pre-amplification-based qPCR methods were applied in quantitative assay of methylation-related genes (TET1,TET2,TET3 and DNMT3A) in mouse early embryonic cells. RESULTS: The sensitivity of these three methods decreased from isothermal pre-amplification-based qPCR method,PCR pre-amplification-based qPCR method to normal qPCR method. The former two methods detected expression of methylation-related genes in a few mouse embryonic cells within a proper PCR cycle number but the PCR pre-amplification-based qPCR method has lower cost than isothermal pre-amplification-based qPCR method. Therefore,we used PCR pre-amplification-based qPCR method to examine methylation-related genes in mouse oocytes and early embryonic cells after 22 h fertilization. The results showed that the expression level of TET1 mRNA was very low and not detectable,the expression of TET2 decreased,and the expression of TET3 and DNMT3A significantly increased (P<0.01) in this process,which was consistent with reported results. CONCLUSION: The sensitivity of isothermal pre-amplification-based qPCR method was the highest among the three qPCR methods. However,the PCR pre-amplification-based qPCR method is the most suitable one for examining multi-gene expression in a few cells in daily practice due to the simple procedure relatively high sensitivity and low expenditure.

Key words: pre-amplification, real time quantitative PCR, mouse, early embryonic cell, methylation-related genes

中图分类号:

Q781

程琳, 孙平楠, 谢庆东, 周小玲. 预扩增qPCR方法检测少量小鼠早期胚胎细胞中DNA甲基化相关基因的表达[J]. 癌变·畸变·突变, 2016, 28(1): 51-55.

CHENG Lin, SUN Pingnan, XIE Qingdong, ZHOU Xiaoling. Detection of expression of methylation-related genes in mouse early embryonic cells using pre-amplification-based real time quantitative PCR[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2016, 28(1): 51-55.

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预扩增qPCR方法检测少量小鼠早期胚胎细胞中DNA甲基化相关基因的表达

Detection of expression of methylation-related genes in mouse early embryonic cells using pre-amplification-based real time quantitative PCR

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