Comparative analysis of diagnostic platforms for measurement of differentially methylated insulin DNA
Main Article Content
Keywords
digital PCR, unmethylated insulin, cell free DNA, type 1 diabetes, diagnostic biomarker
Abstract
Circulating cell-free DNA (cfDNA) has been intensively investigated as a diagnostic and prognostic marker for various cancers. In recent years, presence of unmethylated insulin cfDNA in the circulation has been correlated with pancreatic β-cell death and risk of developing type 1 diabetes. Digital (d)PCR is an increasingly popular method of quantifying insulin cfDNA due to its ability to determine absolute copy numbers, and its increased sensitivity when compared to the more routinely used quantitative PCR. Multiple platforms have been developed to carry out dPCR. However, not all technologies perform comparably, thereby necessitating evaluation of each platform. Here, we compare two dPCR platforms: the QuantStudio 3D (QS3D, Applied Biosystems) and the QX200 (Bio-Rad), to measure copies of unmethylated/methylated insulin plasmids. The QS3D detected greater copy numbers of the plasmids than the QX200 (manual mode), whereas QX200 demonstrated minimal replicate variability, increased throughput, ease of use and the potential for automation. Overall, the performance of QX200, in our hands, was better suited to measure differentially methylated insulin cfDNA.
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References
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2. Francis G, Stein S. Circulating Cell-Free Tumour DNA in the Management of Cancer. Int J Mol Sci. 2015;16(6):14122-42. doi: 10.3390/ijms160614122. PubMed PMID: 26101870; PubMed Central PMCID: PMCPMC4490543.
3. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, et al. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14(9):985-90. doi: 10.1038/nm.1789. PubMed PMID: 18670422; PubMed Central PMCID: PMCPMC2820391.
4. Atkinson MA, Eisenbarth GS, Michels AW. Type 1 diabetes. Lancet. 2014;383(9911):69-82. doi: 10.1016/S0140-6736(13)60591-7. PubMed PMID: 23890997; PubMed Central PMCID: PMCPMC4380133.
5. Herold KC, Usmani-Brown S, Ghazi T, Lebastchi J, Beam CA, Bellin MD, et al. beta cell death and dysfunction during type 1 diabetes development in at-risk individuals. J Clin Invest. 2015;125(3):1163-73. doi: 10.1172/JCI78142. PubMed PMID: 25642774; PubMed Central PMCID: PMCPMC4362259.
6. Husseiny MI, Kaye A, Zebadua E, Kandeel F, Ferreri K. Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death. PLoS One. 2014;9(4):e94591. doi: 10.1371/journal.pone.0094591. PubMed PMID: 24722187; PubMed Central PMCID: PMCPMC3983232.
7. Fisher MM, Watkins RA, Blum J, Evans-Molina C, Chalasani N, DiMeglio LA, et al. Elevations in Circulating Methylated and Unmethylated Preproinsulin DNA in New-Onset Type 1 Diabetes. Diabetes. 2015;64(11):3867-72. doi: 10.2337/db15-0430. PubMed PMID: 26216854; PubMed Central PMCID: PMCPMC4613977.
8. Akirav EM, Lebastchi J, Galvan EM, Henegariu O, Akirav M, Ablamunits V, et al. Detection of beta cell death in diabetes using differentially methylated circulating DNA. Proc Natl Acad Sci U S A. 2011;108(47):19018-23. doi: 10.1073/pnas.1111008108. PubMed PMID: 22074781; PubMed Central PMCID: PMCPMC3223447.
9. Lehmann-Werman R, Neiman D, Zemmour H, Moss J, Magenheim J, Vaknin-Dembinsky A, et al. Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proc Natl Acad Sci U S A. 2016;113(13):E1826-34. doi: 10.1073/pnas.1519286113. PubMed PMID: 26976580; PubMed Central PMCID: PMCPMC4822610.
10. Fisher MM, Perez Chumbiauca CN, Mather KJ, Mirmira RG, Tersey SA. Detection of islet beta-cell death in vivo by multiplex PCR analysis of differentially methylated DNA. Endocrinology. 2013;154(9):3476-81. doi: 10.1210/en.2013-1223. PubMed PMID: 23825129; PubMed Central PMCID: PMCPMC3749470.
11. Pohl G, Shih Ie M. Principle and applications of digital PCR. Expert Rev Mol Diagn. 2004;4(1):41-7. PubMed PMID: 14711348.
12. Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA. Evaluation of digital PCR for absolute DNA quantification. Anal Chem. 2011;83(17):6474-84. doi: 10.1021/ac103230c. PubMed PMID: 21446772.
13. Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A. 1999;96(16):9236-41. PubMed PMID: 10430926; PubMed Central PMCID: PMCPMC17763.
14. Gu J, Zang W, Liu B, Li L, Huang L, Li S, et al. Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study. Oncotarget. 2017;8(40):67810-20. doi: 10.18632/oncotarget.18866. PubMed PMID: 28978074; PubMed Central PMCID: PMCPMC5620214.
15. Allouchery V, Beaussire L, Perdrix A, Sefrioui D, Augusto L, Guillemet C, et al. Circulating ESR1 mutations at the end of aromatase inhibitor adjuvant treatment and after relapse in breast cancer patients. Breast Cancer Res. 2018;20(1):40. Epub 2018/05/18. doi: 10.1186/s13058-018-0968-0. PubMed PMID: 29769099; PubMed Central PMCID: PMCPMC5956618.
16. Selvaraj V, Maheshwari Y, Hajeri S, Chen J, McCollum TG, Yokomi R. Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus". PLoS One. 2018;13(5):e0197184. Epub 2018/05/18. doi: 10.1371/journal.pone.0197184. PubMed PMID: 29772016.
17. Farr RJ, Januszewski AS, Joglekar MV, Liang H, McAulley AK, Hewitt AW, et al. A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy. Sci Rep. 2015;5:10375. Epub 2015/06/04. doi: 10.1038/srep10375
srep10375 [pii]. PubMed PMID: 26035063.
18. Dong L, Meng Y, Sui Z, Wang J, Wu L, Fu B. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material. Sci Rep. 2015;5:13174. doi: 10.1038/srep13174. PubMed PMID: 26302947; PubMed Central PMCID: PMCPMC4548226.
19. Alikian M, Whale AS, Akiki S, Piechocki K, Torrado C, Myint T, et al. RT-qPCR and RT-Digital PCR: A Comparison of Different Platforms for the Evaluation of Residual Disease in Chronic Myeloid Leukemia. Clin Chem. 2017;63(2):525-31. doi: 10.1373/clinchem.2016.262824. PubMed PMID: 27979961.
20. Bosman KJ, Nijhuis M, van Ham PM, Wensing AM, Vervisch K, Vandekerckhove L, et al. Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir. Sci Rep. 2015;5:13811. doi: 10.1038/srep13811. PubMed PMID: 26350506; PubMed Central PMCID: PMCPMC4563360.
21. Lee H, Park YM, We YM, Han DJ, Seo JW, Moon H, et al. Evaluation of Digital PCR as a Technique for Monitoring Acute Rejection in Kidney Transplantation. Genomics Inform. 2017;15(1):2-10. doi: 10.5808/GI.2017.15.1.2. PubMed PMID: 28416944; PubMed Central PMCID: PMCPMC5389945.
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Published
Jun 3, 2019
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Farr RJ, Wong WKM, Maynard C-L, Tersey SA, Mirmira RG, Hardikar AA, Joglekar MV. Comparative analysis of diagnostic platforms for measurement of differentially methylated insulin DNA. J Biol Methods [Internet]. 2019 Jun. 3 [cited 2024 Apr. 25];6(2):e113. Available from: https://jbmethods.org/index.php/jbm/article/view/280
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