Isolation and Extraction of Circulating Tumor DNA from Patients with Small Cell Lung Cancer
- 1 August 2008
- journal article
- Published by Wiley in Annals of the New York Academy of Sciences
- Vol. 1137 (1), 98-107
- https://doi.org/10.1196/annals.1448.020
Abstract
There is no consensus on the optimal protocol for isolation of circulating tumor DNA. We report our comparison of several extraction methods and variables that may affect yield, quality, and contamination of tumor DNA. DNA was extracted from the plasma and serum of five healthy volunteers by means of four different commercially available kits and DNA yield was quantified by real-time PCR. DNA was extracted using the optimum kit from the plasma and serum of an additional 10 healthy volunteers and 10 patients with small cell lung cancer (SCLC) to compare yield and DNA fragment size in plasma versus serum and in those with SCLC versus controls. Time to sample processing was also examined. We found that DNA yield was greatest using the QIAamp Viral Spin Kit. Delayed time to processing led to increased DNA concentrations in serum, but not plasma. The plasma DNA concentration in SCLC patients was significantly higher than in healthy volunteers (24.5 ng/mL versus 5.1 ng/mL, P= 0.002). In contrast, there was no significant difference in serum DNA concentrations between controls and patients that may be explained by the wide variability and range of DNA concentrations in serum. A significantly higher proportion of longer fragments (272 bp/60 bp) was observed in the plasma DNA extracted from patients with SCLC than in healthy controls (13% versus 8%, P= 0.04). There was absence of DNA fragments of 512 bp in healthy control plasma, but faint bands were observed in serum, which is thought to be due to cellular contamination. We conclude that plasma is a more reliable source of tumor DNA then serum and have optimized a robust procedure for plasma tumor DNA isolation that can be applied to translational research studies.Keywords
This publication has 22 references indexed in Scilit:
- Utility of Circulating B-RAF DNA Mutation in Serum for Monitoring Melanoma Patients Receiving BiochemotherapyClinical Cancer Research, 2007
- Detection of mutated BRAFV600E variant in circulating DNA of stage III–IV melanoma patientsInternational Journal of Cancer, 2007
- Detection of Aberrant Methylation of Four Genes in Plasma DNA for the Detection of Breast CancerJournal of Clinical Oncology, 2006
- Increased Integrity of Free Circulating DNA in Sera of Patients with Colorectal or Periampullary Cancer: Direct Quantitative PCR for ALU RepeatsClinical Chemistry, 2006
- Improved Method for Isolating Cell-Free DNAClinical Chemistry, 2005
- P-752 Biomarkers for small cell lung cancer (SCLC): DNA methylationand GD2 synthase transcript levelsLung Cancer, 2005
- Quantification of Circulating DNA in the Plasma and Serum of Cancer PatientsAnnals of the New York Academy of Sciences, 2004
- Preferential Isolation of Fragmented DNA Enhances the Detection of Circulating Mutated k-ras DNAClinical Chemistry, 2004
- Quantification of Free Circulating DNA As a Diagnostic Marker in Lung CancerJournal of Clinical Oncology, 2003
- Plasma DNA microsatellite panel as sensitive and tumor‐specific marker in lung cancer patientsInternational Journal of Cancer, 2003