Tripure isolation reagent

Total RNA was isolated from the three groups (CYGB siRNA (+); CYGB siRNA (−) and control) using TriPure^TM isolation reagent (Roche) according to the manufacturer’s protocol. RNA concentration and purity were measured using a spectrophotometer.

RT-qPCR was performed to investigate the genetic expression of Cxcl1, Cxcl2, Icam1, Vcam1 and clock genes Bmal1, Per1, Cry2, Clock, Rev-Erbα, Rorα, and Sirt1 (Table 1). Gene expression was normalized to that of the house-keeping gene Rplp0. Total RNA was isolated from snap frozen heart fragments using the Tripure^TM Isolation Reagent (Roche, 11667165001) according to the manufacturers’ protocol. After DNAse treatment, 500 ng of total RNA was used for cDNA synthesis using the iScript^TM cDNA synthesis kit (Bio-Rad, 1708891). RT-qPCR were performed using iQ^TM SYBR Green supermix (Bio-Rad, 1708880).

Total RNA was isolated using 1 mL TripureTM Isolation Reagent (Roche) according to the manufacturer's protocol. Following DNAse I (Qiagen) treatment, 500 ng total RNA was used for cDNA synthesis (iScriptTM cDNA synthesis kit, Bio-Rad). qPCR reaction was performed using 10 μL iQTM SYBR Green supermix and 10 μL cDNA. Ribosomal Protein Lateral Stalk Subunit P0 (Rplp0) was selected as the housekeeping gene and was used for calculation of normalised gene expression levels (ΔCt). The sequences of all primers are presented in Supplementary Table S1.

We evaluated the potential effect of the intake of PEB on the expression of three main glucose transporters: GLUT2, GLUT4 and GLUT5. Due to the tissue specificity and pinitol absorption, we measured GLUT2 and GLUT5 in the duodenum, jejunum and ileum, and GLUT4 in the heart. To this end, the aforementioned samples were pulverized in liquid nitrogen and mRNA was isolated using the Tripure^TM Isolation Reagent (Roche Molecular Biochemicals, Pleasanton, CA, USA) according to the manufacturer’s instructions and then gene expression was assessed by real-time PCR analysis; GLUT2 (Rn00563565, Thermo Fisher Scientific, Waltham, MA, USA), GLUT4 (Rn01752377, Thermo Fisher Scientific, Waltham, MA, USA) and GLUT5 (Rn00582000, Thermo Fisher Scientific, Waltham, MA, USA).

RNA was isolated with TriPure^TM isolation reagent (Roche). Cells were separated from the culture media, and 300 uL TriPure^TM was added. After lysis, 60 uL chloroform was added, the mixture was vigorously shaken for 15 seconds and incubated for 3 minutes at room temperature. Next, samples were spun for 15 minutes at 12.000 xg (4°C) and the aqueous phase was mixed with 190 uL isopropanol with 0.44 uL GlycoBlue^TM. After an overnight incubation at −20°C, samples were spun at 12.000 xg for 10 minutes (4°C) and the pellet was 2x washed with 1 mL of 75% ethanol (7.500 xg, 5 minutes, 4°C). Next, the pellet was dried at room temperature for 10 minutes, 18 uL RNase free H2O was added and incubated at 56°C for 10 minutes. RNA concentration was measured with Nanodrop. cDNA synthesis was performed with SensiFAST^TM cDNA Synthesis Kit according to manufacturer's instructions.