Ctnnb1

The 14 candidate reference genes primers and three target genes (ctnnb1, robo4, and notch1) were designed and purchased from Thermo Scientific and were selected in consideration of different intracellular biological function as shown in Table 1. Primer sequence and information are also shown in Table 1. RT-qPCR was performed using Light Cycler®480 in multiwall plate 96. Each 20 μL reaction contained 10μL of 2× Supermix SYBR Green I Master, 1μL forward and reversed primers, 8μL of distilled water, and 1μL of cDNA. The RT-qPCR program was comprised of predenaturation at 95°C for 1 minute, 40 cycles at 95°C for 20 seconds, 60°C for 15 seconds, and 72°C for 15 seconds.
The specificity of the primers and the size of the PCR products were checked using 2% agarose gel electrophoresis and gel-red staining. The threshold values (Cp value) were obtained using a fluorescence threshold of 1.0. Then the results were copied into input file, based on the software requirement. The efficiency of each primer was calculated using the standard gradient [%E = 10^(−1/A)∗100] of RT-qPCR. The standard curve was obtained by plotting the Cp value (y axis) against the logarithm of the total cDNA concentration (x axis).

Complete RNA from the white blood cells (WBC) of the participants was isolated using a FavorPrep Blood/Cultured Cell Total RNA Mini kit (#FABRK001-1, Favorgen Biotech Corporation, Ping-Tung, Taiwan). We targeted RNA-Seq using customized TruSeq Targeted RNA Expression Kits and MiSeq System from Illumina (San Diego, CA, USA). Both the sample preparation and the RNA-Seq procedure were performed pursuant to the manufacturer’s instructions. The customized panel genes used in the TruSeq Targeted RNA Expression are listed in Supplementary Table 3. The quantitative RT-PCR reaction was carried out with an ABI 7500 Fast Real-Time PCR System using the ABI TaqMan Fast Universal PCR master mix. Normalization was determined with human β-actin probe (ACTB, P/N: 4331128, ID: Hs01060665_g1). In order to detect expression level, we obtained the TaqMan probes for β-catenin (CTNNB1, P/N: 4331128, ID: Hs00355049_m1), NFATC1 (P/N: 4331128, ID: Hs00542678_m1), RUNX3 (P/N: 4331128, ID: Hs00231709_m1), ETS1 (P/N: 4331128, ID: Hs00428293_m1), and RARG (P/N: 4331128, ID: Hs01559234_m1) from Thermo Fisher Scientific. We verified the comparative RT-PCR data three times and calculated the fold increase using the comparative 2^-ΔΔCt method.