1 thioglycerol

For RNA isolation, 0.5 cm long colon pieces were mechanically disrupted in a homogenization solution containing 1-Thioglycerol (Promega, Madison, WI, USA) in gentleMACS tissue disrupter tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. RNA was isolated using the Maxwell RSC simplyRNA Tissue Kit (Promega) according to the manufacturer’s instructions. RNA concentration was estimated by measuring absorbance at 260 and 280 nm. Complementary DNA (cDNA) was synthesized using the high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. RT-PCR was performed using FAST qPCR Master Mix and predesigned Taqman assays (Thermo Fisher Scientific) on a QuantStudio 6 system using QuantStudio software (Thermo Fisher Scientific). Mouse Actb was used as endogenous control, measurements were performed in triplicate, and relative expression levels were calculated according to the ΔΔCT method.

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%).