Evaluation of suitable control genes for quantitative polymerase chain reaction analysis of maternal plasma cell-free DNA.

The content stability of commonly used control genes is considered to vary significantly in different independent experimental systems, either in the expression of RNA expression or in the level of DNA content. The present study aimed to examine a panel of six common control genes, including β‑globin (HBB), telomerase (TERT), glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH), albumin (ALB), β‑actin (ACTB) and T cell receptor γ (TRG), in order to evaluate and validate the most reliable control genes for quantitative polymerase chain reaction (qPCR) in investigations for the analysis of fetal‑derived DNA and maternal‑derived DNA in maternal plasma to enable non‑invasive prenatal assessment. Plasma DNA was extracted from the peripheral blood of 20 pregnant femals (gestational age, 18.67 ± 0.58 weeks) using a QIAamp DNA mini kit. Electrophoresis was performed to separate the fetal‑derived DNA and the maternal‑derived DNA at the 300bp position. qPCR was then performed, followed by geNorm‑, NormFinder‑ and BestKeeper‑based analyses to evaluated the content stabilities of the six candidate control genes in the fetal‑derived DNA and maternal‑derived DNA. The subsequent analysis of the experimental data revealed that HBB was expressed in the maternal‑ and fetal‑derived DNA together and in the maternal‑derived DNA alone. In addition, GAPDH in the fetal‑derived DNA enabled efficient normalization for qPCR investigations in the maternal plasma DNA.