Syber green dye

Total RNA extraction, reverse transcription and TaqMan real-time polymerase chain reaction (PCR) for miRNAs were performed according to the manufacturer's instructions as described previously [44 ]. To quantify SIRT1 mRNA, 1 μg of total RNA was reverse-transcribed to cDNA using oligo dT and Thermoscript (TaKaRa), performed using the following conditions: 42°C for 60 min and 85°C for 5 min. SYBER Green Dye (Invitrogen), and specific primers for SIRT1 and GAPDH were used. The sequences of the primers were as follows: SIRT1 (sense): 5′-CTGTTTCCTGTGGGATACCTGACT-3′; SIRT1 (antisense): 5′-ATCGAACATGGCTTGAGGATCT-3′; GAPDH (sense): 5′-GATATTGTTGCCATCAATGAC-3′; GAPDH (antisense): 5′-TTGATTTTGGAGGGATCTC G-3′. The relative amount of SIRT1 mRNA was normalized to GAPDH.

DNA extraction was performed according to the modified technique described by Fernandez Zenoff et al. [33 (link)]. Yeasts were grown in YEPD medium at 28°C under agitation (140 rpm) for 24 h and pelleted by centrifugation at 12,000 rpm. The supernatant was discarded and pellets were incubated for 1 h at 55°C with 0.75 mL of 2% [wt/vol] CTAB isolation buffer (Sigma) (1.4 M NaCl, 0.2% [vol/vol] 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl, pH 8) and sterile glass beads (0.5 mm in diameter, Sigma). Every 15 min, samples were vigorously shaken. After extraction, samples were washed with 0.5 mL chloroform-isoamyl alcohol (24:1, vol/vol) and centrifuged (10 min, 12,000 rpm). DNA was precipitated with 0.5 mL of isopropanol (1 h, 4°C) followed by centrifugation (30 min, 12,000 rpm, 4°C). The pellets were washed twice with 80% cold ethanol, vacuum dried, and dissolved in 50 μL of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). DNA samples were subjected to gel electrophoresis on 0.8% agarose (w/v) using TAE (Tris-acetate-EDTA) 1X as running buffer. Gels were stained with Syber Green dye (Invitrogen). Extracted DNA was stored at −20°C.