Development of certified reference material NMIJ CRM 6205-a for the validation of DNA quantification methods: accurate mass concentrations of 600-bp DNA solutions having artificial sequences

  • Sachie ShibayamaEmail author
  • Shin-ichiro Fujii
  • Kazumi Inagaki
  • Taichi Yamazaki
  • Mariko Yoshioka
  • Satoko Matsukura
  • Akira Sasaki
  • Tetsushi Suyama
  • Naohiro Noda
  • Yuji Sekiguchi
  • Akiko Takatsu
Research Paper


Two 600-bp DNA solutions (DNA600-G and DNA600-T) were developed as certified reference material, NMIJ CRM 6205-a, for the validation of DNA quantification methods. Both DNA600-G and DNA600-T are ideal as “spike-in control” because these materials have artificial nucleic acid sequences. The certified values were determined as the mass concentration of total DNA (whole DNA materials in sample solution regardless of sequence) at 25 °C by formic acid hydrolysis/liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) and inductively coupled plasma-mass spectrometry (ICP-MS) based on the amount of phosphorus. DNAs were synthesized, and plasmids including the synthesized DNAs were cloned into Escherichia coli DH5α. The amplified plasmids were digested with a restriction enzyme and highly purified. Then, the purified DNAs were diluted with water to approximately 1 ng/μL. By using the CRM-validated methods in fields where DNA quantification is required, the reliability of DNA quantification could be improved.

Graphical abstract


DNA quantification Certified reference material Method validation SI traceability LC-IDMS ICP-MS 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2019_1992_MOESM1_ESM.pdf (855 kb)
ESM 1 (PDF 854 kb)


  1. 1.
    Gryson N. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Anal Bioanal Chem. 2010;396:2003–22.CrossRefGoogle Scholar
  2. 2.
    Yoshimura T, Kuribara H, Matsuoka T, Kodama T, Iida M, Watanabe T, et al. Applicability of the quantification of genetically modified organisms to foods processed from maize and soy. J Agr Food Chem. 2005;53:2052–9.CrossRefGoogle Scholar
  3. 3.
    OECD guideline for quality assurance in genetic testing, in. 2007.Google Scholar
  4. 4.
    English CA, Merson S, Keer JT. Use of elemental analysis to determine comparative performance of established DNA quantification methods. Anal Chem. 2006;78:4630–3.CrossRefGoogle Scholar
  5. 5.
    Holden MJ, Haynes RJ, Rabb SA, Satija N, Yang K, Blasic JR. Factors affecting quantification of total DNA by UV spectroscopy and PicoGreen fluorescence. J Agr Food Chem. 2009;57:7221–6.CrossRefGoogle Scholar
  6. 6.
    Mackay IM, Arden KE, Nitsche A. Real-time PCR in virology. Nucleic Acids Res. 2002;30:1292–305.CrossRefGoogle Scholar
  7. 7.
    Wang L, Li PCH. Microfluidic DNA microarray analysis: a review. Anal Chim Acta. 2011;687:12–27.CrossRefGoogle Scholar
  8. 8.
    Nielsen K, Mogensen HS, Hedman J, Niederstaetter H, Parson W, Morling N. Comparison of five DNA quantification methods. Forensic Sci Int-Gen. 2008;2:226–30.CrossRefGoogle Scholar
  9. 9.
    Vogelstein B, Kinzler KW. Digital PCR. P Natl Acad Sci US A. 1999;96:9236–41.CrossRefGoogle Scholar
  10. 10.
    Bhat S, Herrmann J, Armishaw P, Corbisier P, Emslie KR. Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Anal Bioanal Chem. 2009;394:457–67.CrossRefGoogle Scholar
  11. 11.
    Sanders R, Huggett JF, Bushell CA, Cowen S, Scott DJ, Foy CA. Evaluation of digital PCR for absolute DNA quantification. Anal Chem. 2011;83:6474–84.CrossRefGoogle Scholar
  12. 12.
    Whale AS, Cowen S, Foy CA, Huggett JF. Methods for applying accurate digital PCR analysis on low copy DNA samples. PLoS One. 2013;8:e58177.CrossRefGoogle Scholar
  13. 13.
    Dong LH, Meng Y, Wang J, Liu YY. Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers. Anal Bioanal Chem. 2014;406:1701–12.CrossRefGoogle Scholar
  14. 14.
    Corbisier P, Pinheiro L, Mazoua S, Kortekaas AM, Chung PYJ, Gerganova T, et al. DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials. Anal Bioanal Chem. 2015;407:1831–40.CrossRefGoogle Scholar
  15. 15.
    Vynck M, Thas O. Reducing Bias in digital PCR quantification experiments: the importance of appropriately modeling volume variability. Anal Chem. 2018;90:6540–7.CrossRefGoogle Scholar
  16. 16.
    Yoo HB, Park SR, Dong L, Wang J, Sui Z, Pavšič J, et al. International comparison of enumeration-based quantification of DNA copy-concentration using flow cytometric counting and digital polymerase chain reaction. Anal Chem. 2016;88:12169–76.CrossRefGoogle Scholar
  17. 17.
    Haynes RJ, Kline MC, Toman B, Scott C, Wallace P, Butler JM, et al. Standard reference material 2366 for measurement of human cytomegalovirus DNA. J Mol Diagnostics. 2013;15:177–85.CrossRefGoogle Scholar
  18. 18.
    White H, Deprez L, Corbisier P, Hall V, Lin F, Mazoua S, et al. A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR. Leukemia. 2015;29:369–76.CrossRefGoogle Scholar
  19. 19.
    Kawaharasaki M, Suyama T, Arita M, Matsuura N. DNA standard substance, Jp2007–60966, vol. 3; 2007. p. 15.Google Scholar
  20. 20.
    Shibayama S, Fujii S, Takatsu A. HPLC for separation and quantification of deoxyribonucleic acid fragments and measurement of deoxyribonucleic acid degradation. Chromatographia. 2014;77:1333–8.CrossRefGoogle Scholar
  21. 21.
    ISO Guide 35:2006. Reference materials—general and statistical principles for certification. Geneva, 64: International Organization for Standardization (ISO); 2006.Google Scholar
  22. 22.
    Shibayama S, Fujii S, Inagaki K, Yamazaki T, Takatsu A. Formic acid hydrolysis/liquid chromatography isotope dilution mass spectrometry: an accurate method for large DNA quantification. J Chromatogr A. 2016;1468:109–15.CrossRefGoogle Scholar
  23. 23.
    Corbisier P, Vincent S, Schimmel H, Kortekaas AM, Trapmann S, Burns M, et al. CCQM-K86/P113.1: relative quantification of genomic DNA fragments extracted from a biological tissue. Metrologia. 2012;49:8002.CrossRefGoogle Scholar
  24. 24.
    Kinumi T, Narukawa T, Fujii S, Eyama S, Saeki M, Takatsu A. Quantification of an oligonucleotide containing a sequence failure product: comparison of isotope dilution mass spectrometry with other quantification methods. Eur J Mass Spectrom. 2009;15:399–407.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Sachie Shibayama
    • 1
    Email author
  • Shin-ichiro Fujii
    • 1
  • Kazumi Inagaki
    • 1
  • Taichi Yamazaki
    • 1
  • Mariko Yoshioka
    • 1
  • Satoko Matsukura
    • 2
  • Akira Sasaki
    • 2
  • Tetsushi Suyama
    • 2
  • Naohiro Noda
    • 2
  • Yuji Sekiguchi
    • 2
  • Akiko Takatsu
    • 1
  1. 1.National Metrology Institute of Japan (NMIJ)National Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan
  2. 2.Biomedical Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan

Personalised recommendations