MILE Microarray Protocol Validation

There were two stages in the MILE prephase study: protocol training and proficiency testing. As part of the initial protocol training each participating laboratory was provided with identical equipment, including reagent kits, enzymes, spectrophotometer, and heat block instruments, and eight microarray experiments were performed at each centre with an on-site trainer in the respective laboratory being trained. The eight samples analysed during the training course were represented by MCF-7 (breast adenocarcinoma) and HepG2 (liver carcinoma) cell line total RNA (Ambion, Austin, TX, USA) with 1·0 μg and 5·0 μg input of total RNA, respectively, and four leukaemia patient sample lysates prepared from mononuclear cells obtained after Ficoll density purification. Patient lysates comprised cells of one chronic myeloid leukaemia (CML), one chronic lymphocytic leukaemia (CLL), and two replicate lysates of an AML patient sample (containing a translocation t(8;21), French-American-British (FAB) type M2). The total RNA from the patient lysates was extracted at each centre as part of the training programme, making these samples a test of the entire microarray process workflow post sample acquisition (RNeasy kit, Qiagen, Hilden, Germany). Subsequently, after the training phase and for operator proficiency testing, each laboratory independently performed four microarray experiments each for MCF-7 and HepG2 cell lines with inputs of 1·5 μg, 3·0 μg, 5·0 μg, and 8·0 μg total RNA. In total, 204 microarray profiles were included in the analysis (for details see Appendix SI and SII). The three anonymous replicate patient lysates were provided by the Laboratory for Leukaemia Diagnostics in Munich, Germany. All patients gave their informed consent for participation after having been advised of the purpose and investigational nature of the study. The study design adhered to the tenets of the Declaration of Helsinki and was approved by the ethics committees of the participating institutions before its initiation. Details on the microarray analysis workflow, image analysis, quality reports, as well as statistical methods are given in Appendix SI.