P2-063: Clinical and prognostic significance of serum carcinoembryonic antigen level in patients with IIIB and IV stage non-small cell lung cancer: A prospective study

Abstract

Methods: Blood, plasma and serum samples were spiked with known quantities of human DNA. To optimize the yield of DNA, we investigated the effects of changes in the blood collection and processing, storage and DNA extraction protocols. We compared two DNA purification methods, the QIAamp blood DNA kit (QIAGEN) (with and without proteinase K pre-digestion) and phenol/chloroform DNA extraction after heat denaturation (5 minutes at 95˚C). Extracted DNA was quantified using the PicoGreen assay and its quality was checked by conventional PCR analysis for the p53 gene. Results: Using the standard QIAamp protocol, the efficiency of DNA extraction from serum was 13.8 ± 5.9% (Mean ± SD). Pre-incubation with proteinase K (0.4 mg/ml, QIAGEN) at 37˚C for 2 hours significantly increased the DNA recovery (p<0.0001, unpaired t-test), but longer incubation time and incubation at 50˚C did not improve the DNA yield further. Further improvements in the efficiency of the QIAamp protocol were obtained by increasing the volume of elution buffer, but reloading the columns and using different buffers did not improve the yield. Using the phenol/chloroform extraction method after heat denaturation, the efficiency of DNA extraction was 52.7 ± 4.6% (Mean ± SD), which is significantly better than the QIAamp protocol (p<0.0001, unpaired t-test). Delays in processing blood samples reduced the DNA yield. Importantly, all the recovered DNA was of quality suitable for PCR analysis. Conclusions: We recommend that blood samples are held on ice for no more than 1 hour before plasma/serum separation by double spin. Because of its higher efficiency, low-cost and good quality products for PCR analysis, we prefer the phenol/chloroform circulating DNA isolation method with heat denaturation.