Evaluation and validation of the suitable control genes for quantitative PCR studies in plasma DNA for non-invasive prenatal diagnosis

Abstract

Quantitative polymerase chain reaction (qPCR) is widely used in quantitation of plasma DNA for non-invasive prenatal diagnosis (NIPD). Control genes are indispensable as standard normalizers in qPCR analysis, and there is increasing evidence indicating that the content levels of commonly used control genes vary significantly in different independent experiments. The commonly used control genes for DNA quan-titation using qPCR in plasma DNA analysis are frequently chosen without any preliminary evaluation of their suitability. The present study aimed to examine a panel of six common control genes (HBB, TERT, GAPDH, ALB, ACTB and TRG) in order to evaluate and validate the most reliable control genes for qPCR studies in the quantitation of plasma DNA from pregnant and non-pregnant females for NIPD. Plasma DNA was extracted from the peripheral blood of 18 pregnant females and 18 non-pregnant females by the QIAamp DNA mini kit. qPCR followed by geNorm, NormFinder and BestKeeper based analysis was conducted to evaluate the DNA content stabilities of the six candidate control genes. DSCR3 was used to validate the result. The study recommended TERT and the combination of ACTB and TERT as the optimal control genes for qPCR studies on pregnant/non-pregnant plasma DNA quantitation. Thus, the study reveals that the DNA content stability of widely used control genes varies significantly in pregnant and non-pregnant plasma DNA.