Reference Materials: A Golden Criterion in Nucleic Acid Identification

  • Wentao Xu


Identification of nucleic acids needs certificated positive samples for quality control, and reference materials (RMs) are developing to be received by most researchers in molecular biology. However, uniformity and disharmonization exist in the production and regulation of reference materials between different organizations. The objective of this chapter is to review and clarify the definition, attributes and applications, and common regulation process of reference materials in the context of nucleic acid identification.


Reference materials Classification CRMs Biological assessment Quantification 



This work is supported by the National Science and Technology Major Project (2016ZX08012-001). Many thanks to Wenying Tian and Chenguang Wang for their kind help in manuscript conception and preparation.


  1. 1.
    Vesper HW, Thienpont LM. Traceability in laboratory medicine. Clin Chem. 2009;55(6):1067–75.PubMedCrossRefGoogle Scholar
  2. 2.
    Sarton G. Ancient science through the golden age of Greece. Courier Corporation; 2012.Google Scholar
  3. 3.
    Hollihan-Elliot S. Ancient history of China. Mason Crest; 2014.Google Scholar
  4. 4.
    Cochrane RC. Measures for progress: a history of the National Bureau of Standards, vol 275. National Bureau of Standards, US Department of Commerce; 1966.Google Scholar
  5. 5.
    van Lubeck H. Why not replace pH and pOH by just one real acidity grade, AG? J Chem Educ. 1999;76(7):892.CrossRefGoogle Scholar
  6. 6.
    Larsson A, Palm M, Hansson LO, Axelsson O. Reference values for clinical chemistry tests during normal pregnancy. BJOG Int J Obstet Gynaecol. 2008;115(7):874–81.CrossRefGoogle Scholar
  7. 7.
    Guide I. 30 (1992) Terms and definitions used in connection with reference materials. ISO, Geneva; 1992.Google Scholar
  8. 8.
    Rauret G, López-Sánchez J-F, Sahuquillo A, Barahona E, Lachica M, Ure AM, Davidson C, Gomez A, Lück D, Bacon J. Application of a modified BCR sequential extraction (three-step) procedure for the determination of extractable trace metal contents in a sewage sludge amended soil reference material (CRM 483), complemented by a three-year stability study of acetic acid and EDTA extractable metal content. J Environ Monit. 2000;2(3):228–33.PubMedCrossRefGoogle Scholar
  9. 9.
    Sahuquillo A, Lopez-Sanchez J, Rubio R, Rauret G, Thomas R, Davidson C, Ure A. Use of a certified reference material for extractable trace metals to assess sources of uncertainty in the BCR three-stage sequential extraction procedure. Anal Chim Acta. 1999;382(3):317–27.CrossRefGoogle Scholar
  10. 10.
    Kupiec K, Konieczka P, Namieśnik J. Prospects for the production, research and utilization of reference materials. Crit Rev Anal Chem. 2009;39(4):311–22.CrossRefGoogle Scholar
  11. 11.
    Verkouteren RM, Klinedinst DB. Standard reference materials®. NIST Spec Publ. 2004;260:149.Google Scholar
  12. 12.
    Ditmars D, Ishihara S, Chang S, Bernstein G, West E. Enthalpy and heat-capacity standard reference material: synthetic sapphire (a-Al2O3) from 10 to 2250 K. J Res Natl Bur Stand. 1982;87(2):159–63.CrossRefGoogle Scholar
  13. 13.
    Levin BC, Cheng H, Reeder DJ. A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection. Genomics. 1999;55(2):135–46.PubMedCrossRefGoogle Scholar
  14. 14.
    Sharpless KE, Colbert JC, Greenberg RR, Schantz MM, Welch MJ. Recent developments in food-matrix Reference Materials at NIST. Fresenius J Anal Chem. 2001;370(2–3):275–8.PubMedCrossRefGoogle Scholar
  15. 15.
    Blirup‐Jensen S, Grubb A, Lindström V, Schmidt C, Althaus H. Standardization of Cystatin C: development of primary and secondary reference preparations. Scand J Clin Lab Invest. 2008;68(sup241):67–70.CrossRefGoogle Scholar
  16. 16.
    Tate JR, Berg K, Couderc R, Dati F, Kostner GM, Marcovina SM, Rifai N, Sakurabayashi I, Steinmetz A. International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Standardization Project for the Measurement of Lipoprotein (a). Phase 2: selection and properties of a proposed secondary reference material for lipoprotein (a). Clin Chem Lab Med. 1999;37(10):949–58.PubMedCrossRefGoogle Scholar
  17. 17.
    Thorpe S, Koenders A, Group ER. Standardization of steroid receptor assays in human breast cancer—III. Selection of reference material for intra-and inter-laboratory quality control. Eur J Cancer Clin Oncol. 1986;22(8):939–44.PubMedCrossRefGoogle Scholar
  18. 18.
    Klich H, Caliste J. COMAR—database for certified reference materials. Fresenius’ Z Anal Chem. 1988;332(6):552–5.CrossRefGoogle Scholar
  19. 19.
    Steiger T, Pradel R. COMAR 2—the internet database for certified reference materials. Anal Bioanal Chem. 2004;378(5):1145–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Stoeppler M, Wolf WR, Jenks PJ. Reference materials for chemical analysis: certification, availability and proper usage. Wiley; 2008.Google Scholar
  21. 21.
    Quevauviller P. Reference materials: an inquiry into their use and prospects in Europe. TrAC Trends Anal Chem. 1999;18(2):76–85.CrossRefGoogle Scholar
  22. 22.
    Hammes K, Schmidt MW, Smernik RJ, Currie LA, Ball WP, Nguyen TH, Louchouarn P, Houel S, Gustafsson Ö, Elmquist M. Comparison of quantification methods to measure fire‐derived (black/elemental) carbon in soils and sediments using reference materials from soil, water, sediment and the atmosphere. Global Biogeochem Cycles. 2007;21(3):GB3016.CrossRefGoogle Scholar
  23. 23.
    Dulski P. Reference materials for geochemical studies: new analytical data by ICP‐MS and critical discussion of reference values. Geostand Newslett. 2001;25(1):87–125.CrossRefGoogle Scholar
  24. 24.
    Coplen TB, Kendall C, Hopple J. Comparison of stable isotope reference samples. Nature. 1983;302:236–8.CrossRefGoogle Scholar
  25. 25.
    Reynolds BC, Aggarwal J, André L, Baxter D, Beucher C, Brzezinski MA, Engström E, Georg RB, Land M, Leng MJ. An inter-laboratory comparison of Si isotope reference materials. J Anal Atom Spectrom. 2007;22(5):561–8.CrossRefGoogle Scholar
  26. 26.
    Richter S, Alonso A, De Bolle W, Kühn H, Verbruggen A, Wellum R, Taylor P. Re-certification of a series of uranium isotope reference materials: IRMM-183, IRMM-184, IRMM-185, IRMM-186 and IRMM-187. Int J Mass Spectrom. 2005;247(1):37–9.CrossRefGoogle Scholar
  27. 27.
    Steinnes E, Rühling Å, Lippo H, Mäkinen A. Reference materials for large-scale metal deposition surveys. Accred Qual Assur. 1997;2(5):243–9.CrossRefGoogle Scholar
  28. 28.
    Quevauviller P, Rauret G, López-Sánchez J-F, Rubio R, Ure A, Muntau H. Certification of trace metal extractable contents in a sediment reference material (CRM 601) following a three-step sequential extraction procedure. Sci Total Environ. 1997;205(2):223–34.CrossRefGoogle Scholar
  29. 29.
    Eaton DF. Reference materials for fluorescence measurement. Pure Appl Chem. 1988;60(7):1107–14.CrossRefGoogle Scholar
  30. 30.
    Emteborg H, Bordin G. Speciation of organic and inorganic selenium in a biological certified reference material based on microbore ion-exchange chromatography coupled to inductively coupled plasma atomic emission spectrometry via a direct injection nebulizer or coupled to electrothermal atomic absorption spectrometry. Analyst. 1998;123(2):245–53.CrossRefGoogle Scholar
  31. 31.
    Moss D. Accuracy, precision, and quality control of enzyme assays. J Clin Pathol Suppl (Ass Clin Path). 1970;4:22.CrossRefGoogle Scholar
  32. 32.
    Rej R, Vanderlinde RE, Fasce CF. An l-aspartate: 2-oxoglutarate aminotransferase reference material from human erythrocytes: preparation and characterization. Clin Chem. 1972;18(4):374–83.PubMedGoogle Scholar
  33. 33.
    Fasce CF, Rej R, Copeland WH, Vanderlinde RE. A discussion of enzyme reference materials: applications and specifications. Clin Chem. 1973;19(1):5–9.PubMedGoogle Scholar
  34. 34.
    Burtis CA, Sampson EJ, Bayse DD, McKneally SS, Whitner VS. An interlaboratory study of measurement of aspartate aminotransferase activity with use of purified enzyme materials. Clin Chem. 1978;24(6):916–26.PubMedGoogle Scholar
  35. 35.
    Schiele F, Muller J, Colinet E, Siest G, Arzoglou P, Brettschneider H, Calam D, Ceriotti F, Ferard G, Frei J. Interlaboratory study of the IFCC method for alanine aminotransferase performed with use of a partly purified reference material. Clin Chem. 1992;38(12):2365–71.PubMedGoogle Scholar
  36. 36.
    Philipp WJ, van Iwaarden P, Schimmel H, Meeus N, Kollmorgen N. Development of reference materials for microbiological analysis. Accred Qual Assur. 2007;12(3–4):134–8.CrossRefGoogle Scholar
  37. 37.
    Mathieu M, Steghens J, Hørder M, Moss D, Colinet E, Profilis C. The certification of the catalytic concentration of creatine kinase BB in a reconstituted lyophilized material(CRM 299). EUR(Luxembourg); 1992.Google Scholar
  38. 38.
    Duncan P, McKneally S, MacNeil M, Fast D, Bayse D. Development of a reference material for alkaline phosphatase. Clin Chem. 1984;30(1):93–7.PubMedGoogle Scholar
  39. 39.
    Tietz N, Rinker A, Burtis C, Duncan P, Ervin K, Ewen L, Hørder M, Mathieu M, Petitclerc C, Grisley D. Transferability studies for the AACC reference method and the IFCC method for measurement of alkaline phosphatase activity. Clin Chem. 1984;30(5):704–6.PubMedGoogle Scholar
  40. 40.
    Schiele F, Muller J, Colinet E, Siest G. Certification of an enzyme reference material for alkaline phosphatase (CRM 371). Clin Biochem. 1991;24(2):159–68.PubMedCrossRefGoogle Scholar
  41. 41.
    Grubb A, Blirup-Jensen S, Lindström V, Schmidt C, Althaus H, Zegers I. First certified reference material for cystatin C in human serum ERM-DA471/IFCC. Clin Chem Lab Med. 2010;48(11):1619–21.PubMedCrossRefGoogle Scholar
  42. 42.
    Duncan PH, Van Etten RL, MacNeil ML, Shaw LM. Development of a stable reference material for prostatic acid phosphatase. Clin Chem. 1984;30(8):1327–31.PubMedGoogle Scholar
  43. 43.
    Francis J, Moss D, Colinet E, Calam D, Bullock D. A reference preparation of human prostatic acid phosphatase: purification, characterization and field trials. Ann Clin Biochem: Int J Biochem Med. 1992;29(2):176–83.CrossRefGoogle Scholar
  44. 44.
    Hørder M, Rej R, Goldberg D, Werner M. Requirements and functions of reference materials for enzymes. In: Progress in clinical enzymology, vol 2. Masson Paris; 1983. p. 29–34.Google Scholar
  45. 45.
    Quevauviller P. Requirements for production and use of certified reference materials for speciation analysis: a European Commission perspective. Spectrochim Acta B: At Spectrosc. 1998;53(9):1261–79.CrossRefGoogle Scholar
  46. 46.
    Campbell PJ. International biological standards and reference preparations: II. Procedures used for the production of biological standards and reference preparations. J Biol Stand. 1974;2(4):259–67.PubMedCrossRefGoogle Scholar
  47. 47.
    Pauwels J, Lamberty A, Schimmel H. Homogeneity testing of reference materials. Accred Qual Assur. 1998;3(2):51–5.CrossRefGoogle Scholar
  48. 48.
    Linsinger TP, Pauwels J, van der Veen AM, Schimmel H, Lamberty A. Homogeneity and stability of reference materials. Accred Qual Assur. 2001;6(1):20–5.CrossRefGoogle Scholar
  49. 49.
    Lamberty A, Schimmel H, Pauwels J. The study of the stability of reference materials by isochronous measurements. Fresenius J Anal Chem. 1998;360(3–4):359–61.CrossRefGoogle Scholar
  50. 50.
    Wolf WR, Chemistry FoA, Meeting SS. Biological reference materials: availability, uses, and need for validation of nutrient measurement. Wiley; 1985.Google Scholar
  51. 51.
    Minoia C, Sabbioni E, Apostoli P, Pietra R, Pozzoli L, Gallorini M, Nicolaou G, Alessio L, Capodaglio E. Trace element reference values in tissues from inhabitants of the European Community I. A study of 46 elements in urine, blood and serum of Italian subjects. Sci Total Environ. 1990;95:89–105.PubMedCrossRefGoogle Scholar
  52. 52.
    von Behring E. Serum therapy in therapeutics and medical science. Nobel Lectures, Physiology or Medicine 1921; 1901.Google Scholar
  53. 53.
    Kasten FH. Paul Ehrlich: pathfinder in cell biology 1. Chronicle of His life and accomplishments in immunology, cancer research, and chemotherapy 1: Paul Ehrlich’s recipe for success: “Patience, Ability, Money and Luck”. Biotech Histochem. 1996;71(1):2–37.PubMedCrossRefGoogle Scholar
  54. 54.
    Silverstein AM. Paul Ehrlich’s passion: the origins of his receptor immunology. Cell Immunol. 1999;194(2):213–21.PubMedCrossRefGoogle Scholar
  55. 55.
    Kaufmann SH. Immunology’s foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff. Nat Immunol. 2008;9(7):705–12.PubMedCrossRefGoogle Scholar
  56. 56.
    Miller WG, Myers GL, Gantzer ML, Kahn SE, Schönbrunner ER, Thienpont LM, Bunk DM, Christenson RH, Eckfeldt JH, Lo SF. Roadmap for harmonization of clinical laboratory measurement procedures. Clin Chem. 2011;57(8):1108–17.CrossRefGoogle Scholar
  57. 57.
    Einspanier R. Quantifying genetically modified material in food: searching for a reliable certification. Eur Food Res Technol. 2001;213(6):415–6.CrossRefGoogle Scholar
  58. 58.
    Sabbioni E, Minoia C, Pietra R, Fortaner S, Gallorini M, Saltelli A. Trace element reference values in tissues from inhabitants of the European Community. II. Examples of strategy adopted and trace element analysis of blood, lymph nodes and cerebrospinal fluid of Italian subjects. Sci Total Environ. 1992;120(1):39–61.PubMedCrossRefGoogle Scholar
  59. 59.
    Muramatsu Y, Parr R. Survey of currently available reference materials for use in connection with the determination of trace elements in biological and environmental materials. International Atomic Energy Agency Vienna; 1985.Google Scholar
  60. 60.
    Primrose S, Saunders GC, Parkes HC. Analytical molecular biology: quality and validation, vol. 8. Cambridge: Royal Society of Chemistry; 1999.Google Scholar
  61. 61.
    King B. The selection and use of reference materials. A basic guide for laboratories and accreditation bodies, EEE/RM/062rev3; 2002.Google Scholar
  62. 62.
    Commission IE. General Requirements for the Competence of Testing and Calibration Laboratories: Prescriptions Generales Concernant la Competence Des Laboratoires D’etalonnages Et D’essais. International Organization of Standardization; 1999.Google Scholar
  63. 63.
    Janning B, Mooijman K, Havelaar A. Development, production and certification of microbiological reference materials. Fresenius J Anal Chem. 1995;352(1–2):240–5.CrossRefGoogle Scholar
  64. 64.
    Mooijman K, Havelaar A. Application of microbiological (certified) reference materials. Water Sci Technol. 1997;35(11):467–70.CrossRefGoogle Scholar
  65. 65.
    James C. Global status of commercialized biotech/GM crops. Ithaca: International Service for the Acquisition of Agri-biotech Applications (ISAAA); 2010.Google Scholar
  66. 66.
    Zhang D, Guo J. The development and standardization of testing methods for genetically modified organisms and their derived products F. J Integr Plant Biol. 2011;53(7):539–51.PubMedCrossRefGoogle Scholar
  67. 67.
    Li X, Pan L, Li J, Zhang Q, Zhang S, Lv R, Yang L. Establishment and application of event-specific polymerase chain reaction methods for two genetically modified soybean events, A2704-12 and A5547-127. J Agric Food Chem. 2011;59(24):13188–94.PubMedCrossRefGoogle Scholar
  68. 68.
    Zhu X, Chen L, Shen P, Jia J, Zhang D, Yang L. High sensitive detection of Cry1Ab protein using a quantum dot-based fluorescence-linked immunosorbent assay. J Agric Food Chem. 2011;59(6):2184–9.PubMedCrossRefGoogle Scholar
  69. 69.
    Trapmann S, Schimmel H, Kramer GN, Eede G, Pauwels J. Production of certified reference materials for the detection of genetically modified organisms. J AOAC Int. 2002;85(3):775–9.PubMedGoogle Scholar
  70. 70.
    Pi L, Li X, Cao Y, Wang C, Pan L, Yang L. Development and application of a multi-targeting reference plasmid as calibrator for analysis of five genetically modified soybean events. Anal Bioanal Chem. 2015;407(10):2877–86.PubMedCrossRefGoogle Scholar
  71. 71.
    Broothaerts W, Corbisier P, Emons H, Emteborg H, Linsinger TP, Trapmann S. Development of a certified reference material for genetically modified potato with altered starch composition. J Agric Food Chem. 2007;55(12):4728–34.PubMedCrossRefGoogle Scholar
  72. 72.
    Jiang Y, Yang H, Quan S, Liu Y, Shen P, Yang L. Development of certified matrix-based reference material of genetically modified rice event TT51-1 for real-time PCR quantification. Anal Bioanal Chem. 2015;407(22):6731–9.PubMedCrossRefGoogle Scholar
  73. 73.
    Zhang H, Yang L, Guo J, Li X, Jiang L, Zhang D. Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of roundup ready soybean. J Agric Food Chem. 2008;56(14):5514–20.PubMedCrossRefGoogle Scholar
  74. 74.
    Dong W, Yang L, Shen K, Kim B, Kleter GA, Marvin HJ, Guo R, Liang W, Zhang D. GMDD: a database of GMO detection methods. BMC Bioinforma. 2008;9(1):260.CrossRefGoogle Scholar
  75. 75.
    Yu H, Hahn Y, Yang I. Reference materials for calibration of analytical biases in quantification of DNA methylation. PLoS One. 2015;10(9):e0137006.PubMedPubMedCentralCrossRefGoogle Scholar
  76. 76.
    Yoshinaga J, Nakama A, Morita M, Edmonds JS. Fish otolith reference material for quality assurance of chemical analyses. Mar Chem. 2000;69(1):91–7.CrossRefGoogle Scholar
  77. 77.
    Mattiuzzo G, Ashall J, Doris KS, MacLellan-Gibson K, Nicolson C, Wilkinson DE, Harvey R, Almond N, Anderson R, Efstathiou S. Development of lentivirus-based reference materials for Ebola virus nucleic acid amplification technology-based assays. PLoS One. 2015;10(11):e0142751.PubMedPubMedCentralCrossRefGoogle Scholar
  78. 78.
    Stone V, Nowack B, Baun A, van den Brink N, von der Kammer F, Dusinska M, Handy R, Hankin S, Hassellöv M, Joner E. Nanomaterials for environmental studies: classification, reference material issues, and strategies for physico-chemical characterisation. Sci Total Environ. 2010;408(7):1745–54.PubMedCrossRefGoogle Scholar
  79. 79.
    Li XH, Long WG, Li QL, Liu Y, Zheng YF, Yang YH, Chamberlain KR, Wan DF, Guo CH, Wang XC. Penglai zircon megacrysts: a potential new working reference material for microbeam determination of Hf–O isotopes and U–Pb age. Geostand Geoanalytical Res. 2010;34(2):117–34.CrossRefGoogle Scholar
  80. 80.
    Nelson BC, Petersen EJ, Marquis BJ, Atha DH, Elliott JT, Cleveland D, Watson SS, Tseng I-H, Dillon A, Theodore M. NIST gold nanoparticle reference materials do not induce oxidative DNA damage. Nanotoxicology. 2013;7(1):21–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Singapore 2016

Authors and Affiliations

  • Wentao Xu
    • 1
    • 2
  1. 1.Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional EngineeringChina Agricultural UniversityBeijingChina
  2. 2.Beijing Laboratory for Food Quality and Safety, College of Food Science & Nutritional EngineeringChina Agricultural UniversityBeijingChina

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