摘要
目的探讨选择采集和储存血样标本的最佳方案,并讨论不同DNA提取方法的用途方向。方法分别用普通抗凝采血管和DNAgard Blood抗凝采血管采集静脉血5ml,放在室温下2d或-80℃下1周后采用试剂盒法和自配试剂法提取DNA。用核酸定量仪检测纯度和浓度,用凝胶电泳检查完整度,用PCR评估质量和对后续实验的影响。用χ2检验对比不同收集方法、提取方法血样中DNA的效果和质量,并对比用于后续试验的结果。对比工作所消耗的资源和费用。结果二种方法在提取DNA效率上差异无统计学意义;用普通采血管室温放置2d,提取DNA为0;普通采血管-80℃冻存7d和用DNAgardBlood采血管室温放置7d,在DNA提取纯度和提取效率差异均无统计学意义。各种情况下采用二种方法提取的DNA完整性和对PCR试验结果无明显差异。在费用支出和适用强度上,若不计算冰箱储存费用,DNAgard Blood采血管购买价是普通抗凝采血管的10倍;若计算低温保存、运输、采血组织等费用,用DNAgard Blood采血管的总费用是普通采血管费用的50%。结论 DNAgard Blood采血管对全血中DNA有较好的保护作用,实用性好。在有冰箱设备的医院,用普通采血管更容易被接受。
Objective To assess the quality of human blood samples, the impact on the DNA application as- say from blood- samples, and the economic benefits among different methods of blood collection and DNA extraction. Methods The blood samples were collected using the EDTA- Na2 and DNAgard Blood antico- agulant vacuum tubes from volunteer （n = 100） venous blood 5 ml, respectively. A portion of the blood- samples were extracted DNA by using either commercial Kit or self- made reagents after collection in 30 - 60 minutes, which recognized as fresh blood samples. Remaining portions of the blood samples were ex- tracted DNA after stored at either - 80℃ or room temperature （RT） for at least one week （7 days）, which named as storage blood samples. The quality of the extracted DNA was assessed by use of a nucleic acid quantitative instrument to measure the purification and concentration of DNA, the gel electrophoresis to check the integrity of DNA, and the PCR method to verify DNA application assay （a fragment of human vi- tamin D receptor gene, rs2228570）. A chi- square was used to compare the different values of the results between different methods of blood- sample collection, storage, DNA extraction and DNA application as- say, as well as the results of the costs of those processes. The comparison outcomes were described as the 95% confidence of P〈0.05 for significant level. Results All comparisons showed insignificant differences between the two methods of DNA extractions from blood samples with various collections, storages （P〉 0.05）, even in the same result of the null DNA isolation from the blood samples collected by using EDTA- Na2 vacuum tubes and storing at RM for 2 days. Although the similar outcomes of DNA quality from blood - samples either stored in EDTA- Na2 tubes at - 80℃ or in DNAgard Blood tubes at RT for at least one week （7 days） were measured and checked by use a nucleic acid quantitative instrument, gel electrophoresis and DNA application assay, the economical benefits and or costs between use of the two kinds of blood col- lection tubes showed in much different（P〈0.05）. Conclusions The two methods of DNA extraction have their advantages, respectively, as well as advantages and disadvantages showed in the two kinds of blood collection tubes. Given the production characteristics of protect DNA from degradation, the DNAgard blood dcolleetion tubes can be used in more effectively and more extensively research fields. Whereas, the EDTA- Na2 vacuum tube will be easier to be accepted in the well- equipment hospital and clinics for pa- tient diagnosis throμgh blood tests. Therefore, the qualification of the sample collection and storage, and assessment of the experimental methods should be undertaken before the Study commencement accordingly.
出处
《中国煤炭工业医学杂志》
2013年第6期861-866,共6页
Chinese Journal of Coal Industry Medicine
基金
国家自然科学基金(编号:30960339)
