title: 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 quantitation 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.
authors.affiliation: Central Laboratory, Second Hospital, Jilin University, Jilin, Changchun 130041, P.R. China.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Central Laboratory, Second Hospital, Jilin University, Jilin, Changchun 130041, P.R. China.
authors.forename: Hassan Abdellah Ahmed
authors.initials: HA
authors.lastname: Ali
authors.affiliation: Central Laboratory, Second Hospital, Jilin University, Jilin, Changchun 130041, P.R. China.
authors.forename: Shan
authors.initials: S
authors.lastname: Yu
authors.affiliation: Central Laboratory, Second Hospital, Jilin University, Jilin, Changchun 130041, P.R. China.
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: China-Japan Union Hospital, Jilin University, Jilin, Changchun 130033, P.R. China.
authors.forename: Xiuying
authors.initials: X
authors.lastname: Li
authors.affiliation: Central Laboratory, Second Hospital, Jilin University, Jilin, Changchun 130041, P.R. China.
authors.forename: Zhenwu
authors.initials: Z
authors.lastname: Du
authors.affiliation: Central Laboratory, Second Hospital, Jilin University, Jilin, Changchun 130041, P.R. China.
authors.forename: Guizhen
authors.initials: G
authors.lastname: Zhang
mesh_terms: D000199:Actins / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000097:blood / Q000592:standards; D005260:Female; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics; D012015:Reference Standards; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000235:genetics; D000075462:Serum Albumin, Human; D019098:Telomerase / Q000235:genetics; D055544:beta-Globins / Q000235:genetics
chemical_list: C422455:ALB protein, human; D000199:Actins; D016692:Receptors, Antigen, T-Cell, gamma-delta; D012709:Serum Albumin; D055544:beta-Globins; D004247:DNA; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C509186:TERT protein, human; D019098:Telomerase; D000075462:Serum Albumin, Human