Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Apr 1;3(4):921-926.
doi: 10.3892/ol.2012.592. Epub 2012 Feb 3.

Quantitative analysis of cell-free DNA in the plasma of gastric cancer patients

Jong-Lyul Park  1 , Hyun Ja Kim, Bo Youl Choi, Han-Chul Lee, Hay-Ran Jang, Kyu Sang Song, Seung-Moo Noh, Seon-Young Kim, Dong Soo Han, Yong Sung Kim
Affiliations

Quantitative analysis of cell-free DNA in the plasma of gastric cancer patients

Jong-Lyul Park et al. Oncol Lett. .

Abstract

In the present study, an accurate and reproducible method for quantifying cell-free DNA (cfDNA) in human blood was established and tested for its ability to predict gastric cancer in patients. Using 'Alu81-qPCR' to amplify 81-bp Alu DNA sequences, we first estimated the amount of cfDNA in the serum or plasma of 130 patients with gastric cancer to identify which source of cfDNA is more suitable for the biomarker screening of these patients. The results of Alu81-qPCR revealed that the amount of cfDNA in the plasma was low compared with that in the serum, but was found at similar levels among the samples, indicating that the plasma may be a more suitable source of cfDNA for biomarker screening. For the 54 patients with gastric cancer and the 59 age-matched healthy controls, the mean levels of plasma cfDNA were 2.4-fold higher in the patient group compared with the control group, indicating that plasma cfDNA levels may be useful for predicting patients with gastric cancer. The results of our study suggest that Alu81-qPCR is a more reliable method than other techniques, such as the PicoGreen assay, for quantifying cfDNA in human blood, demonstrating the potential to complement current diagnostic procedures for the management of gastric cancer patients.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Quantification of fragmented DNA with Alu81-qPCR analysis. (A) Agarose gel electrophoresis of plasma or serum cfDNA from human blood. DNA (50 ng/lane) was separated on a 1% agarose gel: lanes 1 and 2, plasma cfDNA; lanes 3 and 4, serum cfDNA; M, DNA size marker. (B) Agarose gel electrophoresis of fragmented genomic DNA. Intact genomic DNA was fragmented via sonication for various times (5, 10, 20, 30 and 50 sec). Lane 1, intact genomic DNA; lanes 2, 3, 4, 5 and 6, fragmented DNA sonicated for 5, 10, 20, 30 or 50 sec, respectively. (C) Concentration of fragmented DNA as detected with three assays. Fragmented DNA was quantified in each sample with NanoDrop 1000 using a UV-Vis spectrophotometer (ND-1000), Alu81-qPCR (Alu qPCR) and NanoDrop 3300 using the PicoGreen fluorophore (PicoGreen) in triplicate. Each bar is the mean ± SD. (D) Amplification of the 81-bp Alu fragment from the fragmented DNA. Lane numbers are the same as those of Fig. 1B. cfDNA, cell-free DNA.
Figure 2
Figure 2
Comparison of cfDNA concentration in the serum and plasma of blood from patients with gastric cancer. (A) Concentration of cfDNA in serum and plasma. Serum or plasma cfDNA was simultaneously purified from the blood of patients with gastric cancer and their concentrations were measured using Alu81-qPCR. (B) Correlation between serum and plasma cfDNA concentrations. (C) Change in plasma cfDNA concentration with respect to age. Plasma cfDNA level was plotted against patient age. cfDNA, cell-free DNA.
Table I
Table I
Quantification of plasma cfDNA in patients with gastric cancer with respect to clinicopathological characteristics.
Figure 3
Figure 3
Concentration of plasma cfDNA in the blood of healthy individuals and patients with gastric cancer. (A) Comparison of plasma cfDNA levels in healthy individuals and patients. Concentrations of plasma DNA in patients with gastric cancer were significantly higher compared with those in healthy individuals (P<0.001; Mann-Whitney U test). (B) ROC curve for plasma cfDNA concentrations in healthy individuals and patients with gastric cancer. The area under the ROC curve was estimated using the logistic procedure in SigmaPlot 11.0 software. (C) Concentrations of plasma cfDNA in healthy individuals without gastric cancer. The healthy individuals in Fig. 3A were divided into five subgroups according to endoscopic and pathological observations: normal, individuals with no precancerous lesions; CG, individuals with acute or chronic gastritis; EG, individuals with erosive gastritis; AG, individuals with atrophic gastritis; IM, individuals with intestinal metaplasia. As an individual may have more than one type of precancerous lesions simultaneously, certain individuals were counted more than once in the subgroups. The P-value above the CG box indicates the result from the Mann-Whitney U test vs. the normal group. P-values above the EG, AG and IM boxes show results vs. the normal (top P-value) and CG groups (bottom P-value). The box plot analysis shows the median and the outliers. cfDNA, cell-free DNA; ROC, receiver operating characteristic; AUC, area under the ROC curve.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55:74–108. - PubMed
    1. Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 1977;37:646–650. - PubMed
    1. Fournié GJ, Courtin JP, Laval F. et al. Plasma DNA as a marker of cancerous cell death. Investigations in patients suffering from lung cancer and in nude mice bearing human tumours. Cancer Lett. 1995;91:221–227. - PubMed
    1. Jung K, Stephan C, Lewandowski M. et al. Increased cell-free DNA in plasma of patients with metastatic spread in prostate cancer. Cancer Lett. 2004;205:173–180. - PubMed
    1. Laktionov PP, Tamkovich SN, Rykova EY. et al. Extracellular circulating nucleic acids in human plasma in health and disease. Nucleosides Nucleotides Nucleic Acids. 2004;23:879–883. - PubMed

LinkOut - more resources