Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium.
BACKGROUND
and


OBJECTIVE
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 microg of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 microg 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.


CONCLUSIONS
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.
Rosinger
Silke
S
Division of Endocrinology and Diabetes, Department of Internal Medicine, Ulm University, Ulm, Germany.
Nutland
Sarah
S
Mickelson
Eric
E
Varney
Michael D
MD
Boehm
Bernard O
BO
Olsem
Gary J
GJ
Hansen
John A
JA
Nicholson
Ian
I
Hilner
Joan E
JE
Perdue
Letitia H
LH
Pierce
June J
JJ
Akolkar
Beena
B
Nierras
Concepcion
C
Steffes
Michael W
MW
D001773:Blood Cells; D019411:Clinical Laboratory Techniques / Q000295:instrumentation*; D004247:DNA / Q000032:analysis*; D003922:Diabetes Mellitus, Type 1 / Q000097:blood* / Q000453:epidemiology / Q000235:genetics; D005453:Fluorescence; D014644:Genetic Variation; D005838:Genotype; D014943:Global Health; D006680:HLA Antigens / Q000032:analysis / Q000235:genetics; D006801:Humans; D007391:International Cooperation; D007963:Leukocytes, Mononuclear / Q000276:immunology* / Q000502:physiology; D020641:Polymorphism, Single Nucleotide
D006680:HLA Antigens; D004247:DNA