Szpechcinski A, Struniawska R, Zaleska J, Chabowski M, Orlowski T, Roszkowski K, Chorostowska-Wynimko J
Laboratory of Molecular Diagnostics, National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland.
J Physiol Pharmacol. 2008 Dec;59 Suppl 6:675-81.
In the last decade numerous reports demonstrated that free-circulating DNA in plasma/serum samples might be a promising biomarker in a number of pathologies, including cancer. Thus, choosing the reliable and efficient method of plasma DNA quantification would be an essential step prior to any clinical evaluation of cell-free DNA measurement in cancer patients. The aim of present study was to compare two highly-sensitive DNA quantification methods in regard to their applicability and effectiveness in monitoring the cell-free DNA level in the blood of patients with resectable non-small cell lung cancer. Plasma samples collected from 10 patients before any treatment, after neoadjuvant therapy and subsequent surgery, were used for DNA quantification by direct fluorescent PicoGreen staining and by real-time qPCR in SYBR Green and TaqMan probe approach using beta-actin gene as the amplifying target. The PicoGreen method demonstrated a high level of correlation with both the SYBR Green (r=0.87, P<0.0001) and TaqMan probe approach (r=0.94, P<0.0001). The total DNA content, determined by PicoGreen, proved to be several-fold higher than the amplifiable DNA amount measured by real-time qPCR. Consequently, intercalating fluorochromes, like PicoGreen, might serve as a rapid, accurate, and inexpensive alternative to real-time qPCR for routine dsDNA quantification and multicenter standardization.
在过去十年中，大量报告表明，血浆/血清样本中的游离循环DNA可能是包括癌症在内的多种疾病中有前景的生物标志物。因此，在对癌症患者的游离DNA测量进行任何临床评估之前，选择可靠且高效的血浆DNA定量方法将是至关重要的一步。本研究的目的是比较两种高灵敏度DNA定量方法在监测可切除非小细胞肺癌患者血液中游离DNA水平方面的适用性和有效性。从10例患者在任何治疗前、新辅助治疗后及随后手术前后采集的血浆样本，用于通过直接荧光PicoGreen染色以及使用β-肌动蛋白基因作为扩增靶点的SYBR Green和TaqMan探针法的实时定量PCR进行DNA定量。PicoGreen方法与SYBR Green（r = 0.87，P < 0.0001）和TaqMan探针法（r = 0.94，P < 0.0001）均显示出高度相关性。通过PicoGreen测定的总DNA含量被证明比通过实时定量PCR测量的可扩增DNA量高出几倍。因此，像PicoGreen这样的嵌入荧光染料可能作为实时定量PCR的一种快速、准确且廉价的替代方法，用于常规双链DNA定量和多中心标准化。
