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

Silke Rosinger, Sarah Nutland, Eric Mickelson, Michael D. Varney, Bernard O. Boehm, Gary J. Olsem, John A. Hansen, Ian Nicholson, Joan E. Hilner, Letitia H. Perdue, June J. Pierce, Beena Akolkar, Concepcion Nierras, Michael W. Steffes, T1DGC

Research output: Contribution to journalArticle

12 Citations (Scopus)

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. The Author(s) 2010.

Original languageEnglish (US)
Pages (from-to)S65-S74
JournalClinical Trials
Volume7
Issue number1_suppl
DOIs
StatePublished - Aug 1 2010

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Type 1 Diabetes Mellitus
Blood Cells
DNA
Genotyping Techniques
Transformed Cell Line
HLA Antigens
North America
Human Herpesvirus 4
Genotype
Organizations

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Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA : The Type 1 Diabetes Genetics Consortium. / Rosinger, Silke; Nutland, Sarah; Mickelson, Eric; Varney, Michael D.; Boehm, Bernard O.; Olsem, Gary J.; Hansen, John A.; Nicholson, Ian; Hilner, Joan E.; Perdue, Letitia H.; Pierce, June J.; Akolkar, Beena; Nierras, Concepcion; Steffes, Michael W.; T1DGC.

In: Clinical Trials, Vol. 7, No. 1_suppl, 01.08.2010, p. S65-S74.

Research output: Contribution to journalArticle

Rosinger, S, Nutland, S, Mickelson, E, Varney, MD, Boehm, BO, Olsem, GJ, Hansen, JA, Nicholson, I, Hilner, JE, Perdue, LH, Pierce, JJ, Akolkar, B, Nierras, C, Steffes, MW & T1DGC 2010, 'Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: The Type 1 Diabetes Genetics Consortium', Clinical Trials, vol. 7, no. 1_suppl, pp. S65-S74. https://doi.org/10.1177/1740774510373493
Rosinger, Silke ; Nutland, Sarah ; Mickelson, Eric ; Varney, Michael D. ; Boehm, Bernard O. ; Olsem, Gary J. ; Hansen, John A. ; Nicholson, Ian ; Hilner, Joan E. ; Perdue, Letitia H. ; Pierce, June J. ; Akolkar, Beena ; Nierras, Concepcion ; Steffes, Michael W. ; T1DGC. / Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA : The Type 1 Diabetes Genetics Consortium. In: Clinical Trials. 2010 ; Vol. 7, No. 1_suppl. pp. S65-S74.
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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. The Author(s) 2010.",
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T1 - Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA

T2 - The Type 1 Diabetes Genetics Consortium

AU - Rosinger, Silke

AU - Nutland, Sarah

AU - Mickelson, Eric

AU - Varney, Michael D.

AU - Boehm, Bernard O.

AU - Olsem, Gary J.

AU - Hansen, John A.

AU - Nicholson, Ian

AU - Hilner, Joan E.

AU - Perdue, Letitia H.

AU - Pierce, June J.

AU - Akolkar, Beena

AU - Nierras, Concepcion

AU - Steffes, Michael W.

AU - T1DGC,

PY - 2010/8/1

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N2 - 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. The Author(s) 2010.

AB - 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. The Author(s) 2010.

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