Milea Timbergen

62 RNA Isolation and RT-qPCR Total RNA was extracted from two to four tissue sections (20 µm) of DTF FFPE tumour samples using the RecoverAll TM total nucleic acid isolation kit (Ambion/ Life Technologies) according to the manufacturer’s recommendations. Total RNA quality and concentration were determined using a Nanodrop-2000 (Isogen Life Science). The 260nm/280nm ratio was ≥1.80 for all RNA preparations. The mRNA expression of AXIN2 , CCND1 and DKK1 was assessed by RT-qPCR. In brief: 50 ng RNA was reverse transcribed with RevertAid™ H Minus (Thermo Fischer Scientific, EP0452) to generate cDNA. The resulting 4 μL cDNA was subsequently pre-amplified for 15 cycles in a final volume of 8 μL using Taqman PreAmp mastermix (Thermo Fisher Scientific) in combination with 100-fold diluted primer- probe combinations for AXIN2 , CCND1, Dickkopf 1 ( DKK1 ) and Peptidylprolyl Isomerase A ( PPIA), (Thermo Fisher Scientific; Assay Ids Hs00610344_m1, Hs00765553_m1, Hs00183740_m1 and Cat # 4333763F, respectively). Next, for each sample, four individual - gene transcript specific - real time PCRs were carried out in a Mx3000P (Agilent) for 40 cycles using SensiFast Probe Lo-ROX master mix (BioLine) according the manufacturer’s recommendation and the same four primer-probe combinations. A 10 ng/μL down to 0.04 ng/μL serially diluted RNA sample isolated from MCF7 cells and expressing all targets was used in each experiment to monitor the efficiencies of the RT-qPCRs and a minus RT sample as a negative control. All targets were amplified with an equal efficiency (98%- 110%). Finally, levels of the target genes were normalized on the stable expressed PPIA mRNA levels using the delta Cq normalization method. Statistical analysis of AXIN2, CCND1 and DKK1 expression in WT and CTNNB1 DTF mutants The relative expression values of AXIN2 , CCND1 and DKK1 were measured by RT-PCR on FFPE samples, in the Dutch DTF cohort. Median values and the maximum interquartile range (IQR) were calculated. Because of non-normally distributed data, a Kruskal-Wallis test was performed to identify differences in ranked expression levels of AXIN2, CNND1 and DKK1 (p-value < 0.05, confidence interval 95%) between DTF with various CTNNB1 mutation types (T41A, S45F) and DTF with no mutations in exon 3 of CTNNB1 (WT). A Bonferroni correction was used to correct for multiple testing. To validate the results of the RT-PCR data, the most variable probes of AXIN2, CCND1 and DKK1 , were selected based on the highest IQR using log2 transformed data of the total French cohort. Because of a non-normal distribution, a Kruskal-Wallis test was performed to identify differences in ranks of expression levels of AXIN2, CNND1 and DKK1 (p-value 3