Perfecta sybr green supermix

Venous blood was collected and kept on ice for 45 min. After centrifugation at 2000×g and 4 °C and for 10 min, the supernatant serum was collected and stored in aliquots at − 80 °C until further analysis. Samples with visible haemolysis were excluded. Total small RNAs were extracted from 200 μL serum using the miRNeasy serum plasma isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Synthetic C. elegans (C) miR-39-3p (Qiagen, Hilden, Germany) was spiked-in at a final concentration of 1.6 × 108 copies/μL after the initial denaturation, prior to extraction, to correct the extraction efficiency. The total RNA was eluted in 14 μL of RNase-free water. A fixed volume of 7 μL of eluate was used as input for the cDNA synthesis. RNA was converted to cDNA using the qScript™ microRNA cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD). qPCR was performed with PerfeCta® SYBR® Green Supermix on an Applied Biosystems (Foster City, CA) 7900 Real Time PCR System with the following conditions: 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 15 s and 70 °C for 15 s. MicroRNA expression levels were normalized to the mean of spiked-in miR C-miR-39 and presented as 2^−ΔCt values.

Total RNA was extracted from cultured cells and pulverized frozen tissue by the TRIzol method. For microRNA analysis, all primers were purchased from Quanta BioSciences (Gaithersburg, MD, USA). cDNA was generated using QScript MicroRNA cDNA Synthesis kit (Quanta BioSciences) and q-RT-PCR performed using PerfeCTa SYBR green super mix on a Applied Biosystems 7500 Real-Time PCR Instrument (ThermoFisher), and normalized to RNU6 expression. For mRNA analysis, following column purification using Qiagen RNeasy kit and DNase treatment (Qiagen, Toronto, ON, Canada), cDNA was generated with QScript cDNA super mix (Quanta BioSciences) and analyzed as described above, and normalized to β-actin expression. Primers used were PGC-1α total: Forward 5′-CAGCTTTCTGGGTGGATTGA-3′ and Reverse 5′-GCTCATTGTTGTACTGGTTGGA-3′, Mitofusin-2: Forward 5′-CTCTCAAGCACTTTGTCACTGC-3′ and Reverse 5′-TGTATTCCTGTGGGTGTCTTCA-3′, β-actin: Forward 5′-TTGCTGACAGGATGCAGAAG-3′ and Reverse 5′-TAGAGCCACCAATCCACACA-3′.

RNA was isolated from biological triplicate (cells were grown overnight in separate flasks before harvesting) samples. Equivalent amounts of RNA from input and IP samples (∼350 ng) were used to produce cDNA using random hexadeoxynucleotide primers ([final] = 0.01 μg/μl, Promega #C1181) and AMV reverse transcriptase (0.4 unit/µl) for 1 hr at 42° in a 15 μl volume. Real-time PCR was performed using Quanta PerfeCTa SYBR Green SuperMix in a 96-well plate (in technical triplicate) in an Applied Biosystems StepOnePlus machine. For each input and IP, the average Ct value was calculated from the technical triplicates. Then the %IP was calculated using the equation 2^(Ct_Input(avg)−Ct_IP(avg)). The average and SD of three biological replicates was reported using the AVERAGE and STDEV functions in Microsoft Excel. See File S5 for a list of primers.