Mean Ct values ranged from

8.71 (± 1.31 SD) (18S) across all samples to 26.70 (± 1.69 SD) (TBP). The gene with the lowest standard deviation across all samples was IPO8 which showed an overall SD of 1.28, while the gene with the highest standard deviation across the samples was PGK1 with an overall SD of 2.49. The reference genes displayed a relatively broad range of expression. PGK1 had the widest range of Ct values Ro 61-8048 mw between 8.35 and 29.83 (mean 21.03 ± 2.49 SD, range of 21.47), while B2M had the narrowest range of Ct values between 15.25 and 23.59 (mean 17.10 ± 1.31 SD, range of 8.34). During the subsequent analyses using Statminer Ct values above 36 are excluded and imputed, because Ct values above this level are not reliable. This quality control will thus SP600125 supplier influence the Ct ranges. Table 2 Cycle threshold (Ct) values of candidate reference genes divided in the four tissue

groups. Gene symbol Non-metastatic colon cancer Metastatic colon cancer   Tumour Normal PND-1186 concentration Tumour Normal   Mean SD N Mean SD N Mean SD N Mean SD N 18S 8,095 0,546 18 8,440 1,066 18 8,800 1.066 20 9,408 2,035 20 ACTB 20,003 0,765 18 19,949 1,209 18 20,363 1.209 20 20,578 2,673 20 B2M 17,050 0,996 18 17,041 1,002 18 17,217 1.002 20 17,085 1,632 20 GAPDH 18,503 0,722 18 19,502 1,044 18 19,211 1.044 20 20,145 2,541 20 GUSB 23,274 0,375 18 24,081 0,865 18 23,564 0.865 20 24,060 1,981 20 HMBS 25,328 0,736 18 26,577 0,974 18 25,963 0.974 20 27,030 2,436 20 HPRT1 22,795 0,814 18 24,183 0,750

18 23,320 0.750 20 24,264 1,849 20 IPO8 24,575 0,469 18 25,084 0,780 18 25,099 0.780 20 25,529 2,108 20 PGK1 20,322 1,054 18 21,151 1,012 18 20,996 1.011 20 21,573 3,257 20 POLR2A 24,007 0,634 18 24,508 1,061 18 24,933 1.061 20 25,330 2,590 20 PPIA 17,081 0,485 Carnitine palmitoyltransferase II 18 18,241 0,906 18 17,506 0.906 20 18,335 1,724 20 RPLP0 19,706 0,637 18 20,647 0,952 18 20,319 0.952 20 21,081 2,002 20 TBP 26,157 0,577 18 26,860 1,035 18 26,649 1.035 20 27,110 2,797 20 TFRC 21,774 0,926 18 23,334 1,030 18 22,679 1.030 20 23,663 2,303 20 UBC 21,285 0,675 18 21,771 1,046 18 21,532 1.046 20 22,044 2,180 20 YWHAZ 23,933 0,723 18 25,041 1,275 18 24,457 1.275 20 25,401 2,174 20 Table 3 Cycle threshold (Ct) values of candidate endogenous control genes across all tissue samples.