Lightcycler 480 program

We purified RNA from livers of 10 female and 6 male F1 mice using Trizol (Life Technologies, Grand Island, NY, USA) and synthesized cDNA using 1 μg of RNA and a cDNA synthesis kit (Life Technologies), then diluted the cDNA 20 times and used 3 μl of the diluted cDNA per qPCR reaction. For each gene target, we measured each sample in triplicate using the KAPA Biosystems SYBR Green Mix (Wilmington, MA, USA) in a 12 μl reaction volume and the following conditions: 95°C for 5 minutes, and 50 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. We determined the absolute quantity of each transcript using a standard curve in the Roche Light Cycler 480 program, and normalized the quantity of each sample relative to the quantity of the house-keeping gene Rpl. We compared average levels of each transcript between females and males using a two-tailed t-test.

Total RNA was harvested from heart tissue and cardiomyocytes using Qiazol reagent (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. The concentration of each sample was measured using a Nanodrop ND-2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). For miRNA microarray analysis, miRNA expression profiling was conducted by miRNA microarray analysis using the miRCURY LNA™ miRNA Array (Exiqon, Vedbaek, Denmark) containing 700 mature rat miRNAs. The accuracy of the microarray data was validated using real-time PCR with TaqMan probes. For real-time PCR analysis, total RNA was reverse transcribed with stem-loop primers and the TaqMan® MicroRNA Reverse Transcription kit (Applied BioSystems, Foster City, CA, USA), according to the manufacturer’s instructions [28 (link)]. Real-time PCR was performed in duplicate using the TaqMan® MicroRNA assay kit and TaqMan Universal PCR MasterMix (Applied Biosystems) for miRNA-23a and miRNA-92a, according to the manufacturer’s instructions. Real-time PCR was performed using the LightCycler® 480 program (Roche) for 40 cycles, (10 s each, at 95°C, 60°C, 72°C). Relative miRNA expression levels were normalized using the RNU6B (U6) small non-coding RNA as an endogenous control. The information of TaqMan® MicroRNA assay kit is shown in S1 Table.