Leukemia 2015 Feb;29(2):369-76

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

White H1, Deprez L2, Corbisier P2, Hall V3, Lin F1, Mazoua S2, Trapmann S2, Aggerholm A4, Andrikovics H5, Akiki S6, Barbany G7, Boeckx N8, Bench A9, Catherwood M10, Cayuela JM11, Chudleigh S12, Clench T13, Colomer D14, Daraio F15, Dulucq S16, Farrugia J17, Fletcher L18, Foroni L19, Ganderton R20, Gerrard G19, Gineikienė E21, Hayette S22, El Housni H23, Izzo B24, Jansson M25, Johnels P26, Jurcek T27, Kairisto V28, Kizilors A29, Kim DW30, Lange T31, Lion T32, Polakova KM33, Martinelli G34, McCarron S35, Merle PA36, Milner B37, Mitterbauer-Hohendanner G38, Nagar M39, Nickless G40, Nomdedéu J41, Nymoen DA42, Leibundgut EO43, Ozbek U44, Pajič T45, Pfeifer H46, Preudhomme C47, Raudsepp K48, Romeo G49, Sacha T50, Talmaci R51, Touloumenidou T52, Van der Velden VH53, Waits P54, Wang L55, Wilkinson E56, Wilson G57, Wren D58, Zadro R59, Ziermann J60, Zoi K61, Müller MC62, Hochhaus A60, Schimmel H2, Cross NC1, Emons H2.
Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).
al quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).