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 ß­globin (HBB), telomerase (TERT), glyceraldehyde­3­phosphate dehydrogenase (GAPDH), albumin (ALB), ß­actin (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 fetal­derived DNA and maternal­derived DNA in maternal plasma to enable non­invasive 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 fetal­derived DNA and the maternal­derived DNA at the 300bp position. qPCR was then performed, followed by geNorm­, NormFinder­ and BestKeeper­based analyses to evaluated the content stabilities of the six candidate control genes in the fetal­derived DNA and maternal­derived DNA. The subsequent analysis of the experimental data revealed that HBB was expressed in the maternal­ and fetal­derived DNA together and in the maternal­derived DNA alone. In addition, GAPDH in the fetal­derived DNA enabled efficient normalization for qPCR investigations in the maternal plasma DNA.

Evaluation and validation of the suitable control genes for quantitative PCR studies in plasma DNA for non­invasive prenatal diagnosis.

Quantitative polymerase chain reaction (qPCR) is widely used in quantitation of plasma DNA for non­invasive 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 non­pregnant females for NIPD. Plasma DNA was extracted from the peripheral blood of 18 pregnant females and 18 non­pregnant 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/non­pregnant plasma DNA quantitation. Thus, the study reveals that the DNA content stability of widely used control genes varies significantly in pregnant and non­pregnant plasma DNA.

Recombinant human decorin upregulates p57KIP² expression in HepG2 hepatoma cell lines.

Increasing the expression of cyclin-cyclin-dependent kinase inhibitors (cyclin-CDK) using small molecule inhibitors is a therapeutic strategy used to suppress cancer cell growth. Decorin (DCN), a functional component of the extracellular matrix, has been implicated in the suppression of cell proliferation by upregulating p21, a cyclin-CDK inhibitor. The purpose of this study was to examine the effect of recombinant decorin on the reactivation of p57KIP2, whose expression is silenced in hepatocellular carcinoma (HCC). Cell viability assay, cell cycle analysis, apoptosis assay and quantitative real time-PCR experiments were performed in three groups of HepG2 human cells: Uninfected HepG2 cells (control group), pcDNA3.1 vector-infected HepG2 cells (pcDNA3.1 group) and pcDNA3.1-DCN-infected HepG2 cells (pcDNA3.1­DCN group). Our results revealed that recombinant human decorin inhibited cell proliferation, induced G0/G1 phase arrest and induced apoptosis by increasing the expression of caspase-3 in the pcDNA3.1-DCN group. The expression of p57KIP2 mRNA in the pcDNA3.1-DCN group was higher than in the pcDNA3.1 and control groups (P<0.05); however, there was no statistically significant difference between the control and pcDNA3.1 groups (P>0.05). In conclusion, recombinant human decorin reactivated p57KIP2 expression in HepG2 cells. As the expression level of p57KIP2 is downregulated in HCC, our finding may serve as a basis for the therapy and prognosis of HCC, although further studies are required.