摘要
目的:探讨血细胞样本长期(10年以上)低温保存后获得DNA的质量和产量情况。方法:选取南京总医院国家肾脏疾病临床医学研究中心生物样本库低温(-80℃)保存10年以上3 ml抗凝血分离得到的血细胞样本150份,采用全自动核酸提取仪(磁珠法)提取DNA,Nanodrop 2000测定DNA的纯度和产量,琼脂糖凝胶电泳检查DNA完整性。结果:150份血细胞样本提取的DNAA260/A280平均值为1.84,A260/A230平均值为2.27,产量平均值为187.57μg。所有样本凝胶电泳均可见DNA条带比较集中,未见弥散分布,分子量≥20 kb。150份DNA中,134份1.7≤A260/A280≤1.9,占89.3%;120份A260/A230≥2.0,占80%;115份1.7≤A260/A280≤1.9且A260/A230≥2.0,占76.7%。143份DNA产量≥10μg,占95.3%。其中,DNA产量≥10μg且1.7≤A260/A280≤1.9有132份,占88%。DNA产量≥10μg,1.7≤A260/A280≤1.9且A260/A230≥2.0有114份,占76%;结论:绝大部分低温保存10年以上的血细胞样本磁珠法提取的DNA质量和产量都可满足下游实验要求。该研究为从长期低温保存的血细胞样本中提取DNA用于科学研究提供参考。
Objective：To investigate the quality and yield of DNA extracted from blood cell after long-term（ more than 10 years） storage at-80℃. Methodology：The 150 blood cell samples enrolled in the study were selected from the Renal Biobank of National Clinical Research Center of Kidney Diseases,which were isolated from 3 ml anticoagulant blood having been preserved for more than 10 years. Extraction of DNA was performed on automatic nucleic acid extraction instrument by magnetic beads method.Assay of the purity and yield of DNA by Nanodrop and the integrity by agarose gel electrophoresis. Results： Of the DNA extracted from 150 blood cell sampels,the mean value of A260/A280 is 1. 84,A260/A230 is 2. 27 and yield is 187. 57 μg. Agarose gel electrophoresis showed that all the DNA bands were relatively concentrated without dispersion distribution,and DNA molecular weight larger than 20 kb. In the 150 DNA samples,there are 134 DNA 1. 7≤A260/A280≤1. 9,accounting for 89. 3%,120 DNA A260/A230≥2. 0,accounting for 80%,115 DNA1. 7≤A260/A280≤ 1. 9 and A260/A230 ≥ 2. 0,accounting for 76. 7%. The yield of 143 DNA ≥ 10 μg,accounting for95. 3%.Of which,there are 132 DNA yield≥10 μg and 1. 7 ≤ A260/A280 ≤1. 9,accounting for 88%. Further,114 DNA yield≥10 μg,1. 7 ≤ A260/A280 ≤1. 9,and A260/A230 ≥2. 0,accounting for 76%. Conclusion： DNA extracted from blood cell samples by magnetic beads mostly remains high quality and quantity after long-term cryopreservation in our center,which can meet the demand of downstream experiments. This research offers a reference for selecting samples for scientific research that had been preserved for more than 10 years in Biobank.
作者
窦为娟
卢颖辉
唐蓉
周敏林
贺贝
尹茹
朱萍
曾彩虹
刘志红
郑春霞
DOU Weijuan;LU Yinghui;TANG Rong;ZHOU Minlin;HE Bei;YIN Ru;ZHU Ping;ZENG Caihong;LIU Zhihong;ZHENG Chunxia(National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing University School of Medicine, Nanjing 210016,China)
出处
《肾脏病与透析肾移植杂志》
CAS
CSCD
北大核心
2018年第3期245-249,共5页
Chinese Journal of Nephrology,Dialysis & Transplantation
基金
国家自然科学基金面上项目(81670699)
江苏省创新能力建设专项(BM2015004)
国家重点研发计划项目(2016YFC0904103)
江苏省科技计划项目(BE2016747)
关键词
血细胞
长期
低温保存
DNA
blood cell samples
long-term
cryopreservation
DNA
