Abstract
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 µg of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 µg 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.
Highlights
Type 1 diabetes is a multi-factorial autoimmune disease in which the insulin producing b-cells are selectively destroyed [1]
This was due to use of a contaminated batch of phosphate buffered saline (PBS) buffer purchased as sterile from Inverclyde Biologicals, which later turned out to be nonsterile due to a breach in the sterilization procedure at the factory
The extracted DNA from whole blood cell packs or peripheral blood mononuclear cells (PBMCs) was of high quality and was successfully utilized in the human leukocyte antigen (HLA) genotyping method utilized in Type 1 Diabetes Genetics Consortium (T1DGC) [8]
Summary
Type 1 diabetes is a multi-factorial autoimmune disease in which the insulin producing b-cells are selectively destroyed [1]. The etiology of type 1 diabetes is only partially characterized; it is generally accepted that a certain genetic predisposition as well as environmental impacts [2] increase the risk to develop the disease. The Type 1 Diabetes Genetics Consortium (T1DGC) brought together several groups of investigators worldwide who shared the common goal of identifying genes related to the etiologies of type 1 diabetes. With the advent of high-throughput instrumentation to process the DNA for its genetic information, more research groups propose utilizing the DNA from clinical studies and trials. The T1DGC repositories extracted DNA and provided the samples to laboratories for human leukocyte antigen (HLA) typing and genotyping. The processes and protocols underlying efforts of the T1DGC to provide adequate amounts of high-quality DNA are described in this article
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