Nanodrop software

To determine alterations in mRNA expression levels after exercise in AKIP1-TG mice, qRT-PCR was performed. Total RNA was isolated from snap frozen LV tissue using Trizol reagent (Invitrogen Corporation, USA), as performed before^{19 (link)}. Using Nanodrop software (ThermoFisher, USA), RNA concentrations were quantified and tested for purity. Next, equal RNA quantities (500 ng) were reverse transcribed to cDNA with Quantitect Reverse Transcription kit (Qiagen, the Netherlands, no. 205313). cDNA was amplified with qRT-PCR for specific primers, as presented in Supplementary Table S1. Standard running protocol was performed: 3 min at 95 °C, followed by 35 cycles of (1) 15 s at 95 °C, (2) 30 s at 60 °C; followed by a dissociation step and melting steps (Bio-Rad CFX384, USA). Results were analysed using the ddCT method normalizing for housekeeping gene 36b4 and WT Sed control group.

A quantum fluorometer (Promega, Madison, WI, USA) was utilized to measure the concentration of extracted nucleic acids; preprogrammed settings were available for quantitating DNA (QuantiFluor ds DNA, Promega) and RNA (QuantiFluor RNA, Promega). A volume (100 μL) of diluted nucleic acid sample (1 μL of nucleic acid + 99 μL buffer) was mixed with the appropriate QuantiFluor dye. After 5 minutes of incubation at room temperature, the DNA or RNA concentrations were determined in the fluorometer. The nucleic acid samples were also read in a spectrophotometer, equipped with Nanodrop software (ThermoFisher, Waltham, MA, USA), at 260 nm and 280 nm. If the results were between 1.7 and 1.9, the samples were considered to be contamination free and adequate for further analyses.

1 x 10⁸ cells were harvested at 800 x g for 10 min at 4°C and washed with ice-cold PBS and quick-frozen in dry ice for 1 min. RNA was purified using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. RNA concentration was quantified using an ND-1000 spectrophotometer and Nanodrop software (Nanodrop Technologies). cDNA synthesis and qRT-PCR reaction setup was performed as described previously [56 (link)]. qRT-PCR was performed using iQ-SYBRGreen Supermix on a MiniOpticon Real-Time PCR Detection System (Bio-Rad) and was quantified using Bio-Rad CFX Manager software (Bio-Rad). The following primers were used for qRT-PCR: bTub-RTF (5’-CAAGATGGCTGTCACCTTCA-3’), bTub-RTR (5’-GCCAGTGTACCAGTGCAAGA-3’); USP1 RTF (5’-GAGATGGCACCATCACTCCT-3’), USP1 RTR (5’-GTGGGCAGCACCTCTAGAAC-3’); VDU1 RTF (5’-GTCGAAAGACGTGTGGGTTT-3’), VDU1 RTR (5’-GGAGCGAGGGAAGAGAGATT-3’); ISG65-RTF (5’-GAGCATGTTGATAGAGGGATTG-3’), ISG65-RTR (5’-CATTGCTGTTCTCTGATGTCTG-3’); ISG75-RTF (5’-GAGGGCAGCGAGGCCAAG-3’), ISG75-RTR (5’-CTTCCTACGGCCCCTAATAAC-3’).