Journal of Jilin University Medicine Edition ›› 2016, Vol. 42 ›› Issue (03): 523-529.doi: 10.13481/j.1671-587x.20160320

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Evaluation on stability of copy number of reference gene in plasma DNA of pregnant woman

YANG Qiwei, DU Zhenwu, YU Shan, GAO Sujie, ZHAO Guanjie, ZHANG Lin, LU Jia, REN Ming, ZHANG Guizhen   

  1. Central Laboratory, Second Hospital, Jilin University, Changchun 130041, China
  • Received:2015-09-29 Published:2016-06-17

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

CLC Number: 

  • R715.5