Research and Development Division, Streck Inc., Omaha, NE 68128, USA.
Clin Biochem. 2013 Oct;46(15):1561-5. doi: 10.1016/j.clinbiochem.2013.06.002. Epub 2013 Jun 13.
To study the ability of a stabilizing reagent to prevent cellular DNA contamination of cell-free DNA (cfDNA) in plasma during whole blood sample storage and shipping.
Samples were drawn from healthy donors into K₃EDTA and Cell-Free DNA BCTs (BCT) and stored at room temperature (RT). Aliquots were removed at specified time points and cfDNA was purified from the plasma. A Droplet Digital PCR (ddPCR) assay that amplifies a short β-actin gene fragment (136 bp) was used to measure the total plasma cfDNA (pDNA) concentration while a longer β-actin fragment (420 bp) was used to quantify genomic DNA (gDNA). In a follow-up experiment, blood samples drawn into the same types of tubes were shipped round trip by overnight air before cfDNA was isolated and analyzed.
Blood stored in K₃EDTA tubes at RT showed increases in pDNA and gDNA concentrations over time. However, both pDNA and gDNA levels remained stable in BCT for at least seven days. On day 14, there was a 4.5-fold increase in pDNA in BCT as compared to >200-fold increase in K₃EDTA tubes. Likewise, gDNA increased <2-fold on day 14 in BCT as opposed to a 456-fold increase in K₃EDTA tubes. Similar results were observed after samples were shipped.
Cell-Free DNA BCTs prevent gDNA contamination that may occur due to nucleated cell disruption during sample storage and shipping. This novel blood collection tube provides a method for obtaining stable cfDNA samples for rare target detection and accurate analysis while mitigating the threat of gDNA contamination.
研究一种稳定剂在全血样本储存和运输过程中防止游离 DNA（cfDNA）细胞内 DNA 污染的能力。
从健康供体中抽取样本至 K₃EDTA 和游离 DNA BCT（BCT）管中，并在室温下储存。在规定的时间点取出等分试样，并从血浆中纯化 cfDNA。使用扩增短β-肌动蛋白基因片段（136bp）的液滴数字 PCR（ddPCR）测定法测量总血浆 cfDNA（pDNA）浓度，而较长的β-肌动蛋白片段（420bp）则用于定量基因组 DNA（gDNA）。在后续实验中，将相同类型的管中的血液样本通过隔夜空运来回运输，然后分离和分析 cfDNA。
在 RT 下储存在 K₃EDTA 管中的血液随时间推移 pDNA 和 gDNA 浓度增加。然而，BCT 中 pDNA 和 gDNA 水平至少在七天内保持稳定。在 BCT 中，与 K₃EDTA 管中 >200 倍的增加相比，第 14 天 pDNA 增加了 4.5 倍。同样，BCT 中 gDNA 在第 14 天增加了 <2 倍，而 K₃EDTA 管中增加了 456 倍。在样本运输后也观察到了类似的结果。
游离 DNA BCT 可防止因样本储存和运输过程中核细胞破裂而导致的 gDNA 污染。这种新型血液采集管为获取稳定的 cfDNA 样本以进行稀有靶标检测和准确分析提供了一种方法，同时减轻了 gDNA 污染的威胁。
