Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium
Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium
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DOI：
10.1177/1740774510373493
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发表时间：
2010-08-01
期刊：
影响因子：
2.7
通讯作者：
Steffes, Michael W.
中科院分区：
文献类型：
--
作者：
Rosinger, Silke;Nutland, Sarah;Steffes, Michael W.
背景和目的为了获得大量的DNA用于基因型分析和提供可再生的DNA来源，1型糖尿病遗传学联盟（T1 DGC）从世界几个地区的1型糖尿病患者及其家庭成员中收集DNA和外周血单核细胞（PBMC）。为了解决区域特定的需求，不同的方法和样品处理技术之间的实验室提取和定量DNA，并建立EB病毒转化的细胞line.Results超过98%的PBMCs样品被成功转化。每毫升全血提取约20-25 μ g DNA。从细胞包中提取的DNA范围为每个细胞包92至165 μ g。此外，从全血或转化细胞中提取的DNA被成功地用于每个区域人类白细胞抗原基因分型实验室和几个进行联合体范围基因分型项目的其他实验室。DNA的测量很难协调。结论DNA储存库可以在世界不同地区建立，并产生类似数量的DNA。高质量的DNA用于各种高通量基因分型技术。此外，即使运输所需的距离和时间，PBMC的高效转化也是可能的。对于涉及不同地点多个实验室的未来研究/试验，T1 DGC经验包括可能适用的方案示例。总而言之，T1 DGC已经开发出了一些方案，这些方案将对任何试图克服与跨越位于世界不同地区的多个研究设施的研究/试验相关的后勤问题的科学组织感兴趣。临床试验2010; 7：S65-S74。http://ctj.sagepub.com
Background and Purpose To yield large amounts of DNA for many genotype analyses and to provide a renewable source of DNA, the Type 1 Diabetes Genetics Consortium (T1DGC) harvested DNA and peripheral blood mononuclear cells (PBMCs) from individuals with type 1 diabetes and their family members in several regions of the world.Methods DNA repositories were established in Asia-Pacific, Europe, North America, and the United Kingdom. To address region-specific needs, different methods and sample processing techniques were used among the laboratories to extract and to quantify DNA and to establish Epstein-Barr virus transformed cell lines.Results More than 98% of the samples of PBMCs were successfully transformed. Approximately 20-25 mu g of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 mu g per cell pack. In addition, the extracted DNA from whole blood or transformed cells was successfully utilized in each regional human leukocyte antigen genotyping laboratory and by several additional laboratories performing consortium-wide genotyping projects.Limitations Although the isolation of PBMCs was consistent among sites, the measurement of DNA was difficult to harmonize.Conclusions DNA repositories can be established in different regions of the world and produce similar amounts of high-quality DNA for a variety of high-throughput genotyping techniques. Furthermore, even with the distances and time necessary for transportation, highly efficient transformation of PBMCs is possible. For future studies/trials involving several laboratories in different locations, the T1DGC experience includes examples of protocols that may be applicable. In summary, T1DGC has developed protocols that would be of interest to any scientific organization attempting to overcome the logistical problems associated with studies/trials spanning multiple research facilities, located in different regions of the world. Clinical Trials 2010; 7: S65-S74. http://ctj.sagepub.com