孕妇血浆DNA中内参基因拷贝数稳定性的评价

doi:10.13481/j.1671-587x.20160320

摘要/Abstract

摘要：

目的: 探讨孕妇血浆和非孕妇血浆游离DNA中常用内参基因的拷贝数稳定性,为孕妇血浆游离DNA的定量研究提供参考。方法: 选择孕龄为12周左右的健康孕妇及健康未孕女性各18名,取外周静脉血并提取血浆DNA,将DNA样本分为孕妇与非孕妇整体组、孕妇组、非孕妇组、母体与胎儿整体组、母源DNA组和胎源DNA组,选取β-珠蛋白(HBB)、端粒酶(TERT)、甘油醛-3-磷酸脱氢酶(GAPDH)、白蛋白(ALB)、β-肌动蛋白(ACTB)和T细胞受体γ(TRG)为内参基因。采用实时荧光定量PCR(qPCR)法分析血浆DNA中内参基因Ct值,分别采用3款统计学软件geNorm、NormFinder和BestKeeper比较6种内参基因在各组样本中的拷贝数稳定性。结果: 1qPCR法,6种内参基因的所有PCR产物均特异性扩增。各组内参基因Ct值比较差异均无统计学意义(P>0.05)。各组ACTB的Ct值最低,HBB次之,即血浆DNA中ACTB和HBB拷贝数最高。2按照geNorm软件计算结果基因稳定值(M)由高到低的顺序、NormFinder软件计算结果含量稳定性由高到低的顺序、BestKeeper软件计算结果相关系数(R)由高到低的顺序,在各组样本中将6种内参基因按照其拷贝数稳定性由高到低进行排序。综合分析3种软件的分析结果,在孕妇与非孕妇整体组、孕妇组、非孕妇组、母体与胎儿整体组、母源DNA组和胎源DNA组中,拷贝数稳定性最高的内参基因分别为TERT、ACTB、ALB、HBB、HBB及HBB。3母体与胎儿整体组、母源DNA组、母源DNA组和胎源DNA组内参基因的Ct值比较差异均有统计学意义(F=114.84,P<0.05)。结论: 当采用qPCR法对来自于孕妇与非孕妇整体、孕妇、非孕妇、母体与胎儿整体、母源DNA和胎源DNA的血浆游离DNA进行定量分析时,推荐分别选择TERT、ACTB、ALB、HBB、HBB及HBB作为校正内参基因。

关键词: 实时荧光定量PCR, 内参基因, 孕妇, 血浆DNA

Abstract:

Objective: To investigate the stabilities of copy number of common reference genes in cell-free DNA in plasma of the pregnant women and non-pregnant women,and to provide the reference for the quantitative study of cell-free DNA in plasma of the pregnant women. Methods: Eighteen healthy pregnant women with twelve weeks of gestation and eighteen healthy non-pregnant women were investigated.The plasma cell-free DNA was extracted from the peripheral blood.All the DNA samples were divided into pregnant+non-pregnant group,pregnant group,non-pregnant group,maternal+fetal group,maternal group,and fetal group.The HBB,TERT,GAPDH,ALB,ACTB,and TRG were chosen as the reference genes.The Ct values of six reference genes in various groups were obtained by Real-time fluoresence quantitative PCR (qPCR) method.Three statistical softwares (geNorm,NormFinder and BestKeeper) were applied for evaluating the stabilities of copy number of six reference genes in various groups. Results: 1PCR products of all six kinds of reference genes were specific amplification by qPCR method.The differences of Ct values of reference genes in various groups were not statistically significant (P>0.05).The Ct value of ACTB in each group was the lowest,and followed by HBB,which meaned the ACTB and HBB had the highest copy number in the plasma cell-free DNA.2According to the descending order of average expression stability value (M) calculated by geNorm software,the descending order of stability value calculated by NormFinder software,and the descending order of coefficient of correlation value (R) calculated by BestKeeper software,the copy number stabilities of six reference genes were sorted in descending order in various groups.Comprehensive analysis of the results from three softwares,in pregnant+non-pregnant group,pregnant group,non-pregnant group,maternal+fetal group,maternal group, and fetal group,the genes with the highest copy number stabilities were TERT,ACTB,ALB,HBB,HBB,and HBB,respectively.3The differences of Ct values of reference genes between maternal+fetal group, maternal group,maternal group and fetal group were statistically significant (F=114.84,P<0.05). Conclusion: When performing quantitative analysis of plasma cell-free DNA of subjects in pregnant+non-pregnant group,pregnant group,non-pregnant group,maternal+fetal group,maternal group, and fetal group through qPCR method,TERT,ACTB,ALB,HBB,HBB and HBB are recommended respectively as the calibration reference genes.

Key words: Real-time fluorescence quantitative PCR, reference gene, pregnant woman, plasma DNA

中图分类号:

R715.5

杨麒巍, 杜珍武, 于杉, 高素洁, 赵冠杰, 张琳, 卢佳, 任明, 张桂珍. 孕妇血浆DNA中内参基因拷贝数稳定性的评价[J]. 吉林大学学报(医学版), 2016, 42(03): 523-529.

YANG Qiwei, DU Zhenwu, YU Shan, GAO Sujie, ZHAO Guanjie, ZHANG Lin, LU Jia, REN Ming, ZHANG Guizhen. Evaluation on stability of copy number of reference gene in plasma DNA of pregnant woman[J]. Journal of Jilin University Medicine Edition, 2016, 42(03): 523-529.

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