Homogenization solution

For DNA extraction, tissues were initially homogenized in a homogenization solution (Promega) and then processed with Maxwell® RSC Tissue DNA Kit (Promega). Before loading samples onto Maxwell® RSC Cartridges, 300 μl of Lysis Buffer and 30 μl of Proteinase K were added to the homogenized samples, and further incubated for 20 min at 56°C. DNA was quantified with Qubit™ 4 fluorometer using Qubit™ dsDNA High Sensitivity Assay Kit (Invitrogen).

We aimed to measure variation in assay performance over time for individual laboratories and assess how this correlated with stability of CFs. We prepared high and low internal quality control standards by making mixtures of HL60 and K562 cell lines (see Supplementary Information) which were stored and distributed as lysates in either Trizol (Thermo Fisher Scientific), RLT (QIAGEN), or Homogenization Solution containing 1-Thioglycerol (Promega). These standards had BCR::ABL1^IS values of approximately 5% (high level control) and 0.05% (low level control). Participants were asked to use their established protocols to extract RNA from both controls on a monthly basis, prepare two independent cDNA samples and test by RT-qPCR. Each laboratory submitted a minimum of 12 results from both high- and low-level controls over the 6-month period of the study. Data were submitted for reference gene transcript number, BCR::ABL1 transcript number, %BCR::ABL1/reference gene and BCR::ABL1^IS for each IQC sample type. Six batches of high- and low-level control samples were distributed to 46 laboratories and 43 data sets were returned from 41 laboratories at the completion of the study (89%).