Development of a Robust and User-Friendly Method for Leukemia Classification Using Microarrays.

Gene expression profiling (GEP) is a powerful technology for the molecular analysis of leukemia and it groups biologically defined disease entities into distinct sub-classes that can provide diagnosis, guide therapy, and even correlate with disease prognosis. The experimental procedures of micorarray analysis are often cumbersome and provide ample opportunity for variability in gene expression data. We previously reported on our efforts to standardize micorarray analysis across 11 participating laboratories within the international MILE study (Microarray Innovations in LEukemia) where a large dataset of over 4,000 leukemia patient samples is being generated using both Affymetrix HG-U133 Plus 2.0 and custom format microarrays. For a better applicability in a routine laboratory workflow and in order to improve the robustness of the micorarray analysis we now have modified the original micorarray sample preparation protocols as originally published by the manufacturer. Here we report the final results of this effort to minimize the complexity of the sample preparation protocol and to reduce the time that is necessary to run the assay. We designed pre-assembled kits for total RNA preparation, nucleic acid cleanup, cDNA synthesis, in vitro transcription, hybridization and staining, and wash buffers guiding the operator through the whole process of sample preparation to microarray result generation. To further improve the ease of use of this assay we minimized to a large extent the overall complexity of sample amplification and labeling, as well as target hybridization and detection procedures. For example, for the RNA amplification, cRNA labeling, and signal detection process, the number of individual reagent vials was reduced from 32 to 13 vials. This was achieved by combining individual components to ready-to-use master mixes. Furthermore, starting from total RNA, the time required for generation of labeled and fragmented cRNA has been reduced to a convenient eight hour work-shift. Overall, compared to the original 48 hour protocol as recommended by the manufacturer the new workflow generates microarray data in 26 hours. In total, this development program included n=900 whole genome microarray tests. By comparison testing of the original and the final modified protocols on we further can demonstrate by squared correlation coefficients both high inter-assay (R² > 0.9) and intra-assay (R² > 0.9) reproducibility and precision of gene expression data of this new sample preparation method. Data from cell lines, normal bone marrow, as well as leukemia samples representing the subclasses AML with normal karyotype or other abnormalities, AML with complex aberrant karyotype, CML, and CLL indicate that reproducible subclassification of leukemias is feasible as all samples were predicted by a classification algorithm as the same class as when the samples were prepared according to the the original method. In conclusion, we developed a robust sample processing methodology for microarray analysis of leukemia samples that allows to generate standardized and reproducible microarray results in multiple laboratories.