Ultrapure pcr grade water

Total RNA was extracted from C2C12 cells using RNeasy mini columns (QIAGEN) according to the manufacturer's guidelines. RNA was converted into cDNA with the High‐Capacity cDNA Kit (Applied Biosystems) according to the manufacturer's guidelines using oligo (dT) and Moloney murine leukemia virus reverse transcriptase. The cDNA was used for quantitative PCR analysis, carried out on a StepOnePlus PCR system (Applied Biosystems) using Absolute Blue QPCR Master Mix (Thermo Scientific) with SYBR Green. The reaction protocol was as follows: 15 min at 95°C for enzyme activation, followed by 40 cycles of 15 s at 95°C, 30 s at 60°C, and 15 s at 72°C, at the end of which fluorescence was measured with the Rotor‐Gene PCR Cycler. SYBR Green I assays also included a melting curve at the end of the cycling protocol, with continuous fluorescence measurement from 65–99°C. All reactions contained the same amount of cDNA, 10 μl Absolute Blue QPCR Master Mix, primers for the indicated genes (Table 1) and UltraPure PCR‐grade water (Biological Industries) to a final volume of 20 µl. Each real‐time PCR included a no‐template control, in duplicate. Relative expression levels (ΔΔCt) were calculated by normalizing to hypoxanthine‐guanine phosphoribosyltransferase (HPRT). Primers were designed using the primer3 website (http://frodo.wi.mi‐ t.edu/primer3).

All real-time PCRs were carried out on a step one plus (ThermoFisher Scientific, Waltham, MS), using the Absolute Blue QPCR Master Mix (ThermoFisher Scientific, Waltham, MS) with SYBR Green. The following is the reaction protocol: 15 min at 95 °C for enzyme activation, followed by 40 cycles of: 15 s at 95 °C, 30 s at 60 °C, and 15 s at 72 °C, at the end of which fluorescence was measured with the Rotor-Gene. SYBR Green-I assays also included a melt curve at the end of the cycling protocol, with continuous fluorescence measurement from 65 to 99 °C. All reactions contained the same amount of cDNA, 10 μl Absolute Blue QPCR Master Mix, primers for the indicated genes (Additional file 2: Table S1) and UltraPure PCR-grade water (Biological Industries, Israel) to a final volume of 20 μl. Each real-time PCR included a no-template control, in duplicate. Relative expression levels (ΔΔCt) were calculated by normalizing to hypoxanthine guanine phosphoribosyl transferase (HPRT). Primers were designed using the primer3 website (http://bioinfo.ut.ee/primer3-0.4.0/).