Tripure extraction reagent

The expression of inducible nitric oxide synthase (iNOS) and arginase1 (Arg-1) in the atria was measured by real-time PCR. Total RNA was isolated from atrial samples using Tripure extraction reagent (ELK Biotechnology, China) in accordance with the manufacturer's protocol. cDNA was synthesized using an EntiLink™ first-strand cDNA synthesis kit (ELK Biotechnology, China). Real-time fluorescent quantitative PCR was performed using a StepOne real-time PCR instrument (Life Technologies, Gaithersburg, MD). Each sample was measured in triplicate using an EnTurbo™ SYBR Green PCR SuperMix kit (ELK Biotechnology, China). Primers used for RT-PCR were: Canine iNOS: 5′-ACCAATACAGGCTCGTGCAG-3′ (forward), 5′-GGGCTGTCTACTACTCGCTCC-3′ (reverse); Canine GAPDH: 5′-GAAGGTCGGAGTGAACGGATT-3′ (forward), 5′-CATTTGATGTTGGCGGGATC-3′ (reverse); Canine Arg-1: 5′-GGCAGAAGTCAAGAAGAACGG-3′ (forward), 5′-CTTTGGCAGATAGGCAAGGAG-3′ (reverse); Canine GAPDH: 5′-GAAGGTCGGAGTGAACGGATT-3′ (forward), 5′-CATTTGATGTTGGCGGGATC-3′ (reverse).

Total RNA was extracted from exosomes, BMSCs induced for 14 days or callus tissues harvested from fracture regions at 21 days after surgery using TRIpure Extraction Reagent (EP013, ELK Biotechnology). The cDNA was reverse transcribed using EntiLink™ 1st Strand cDNA Synthesis Kit (Eq. 003, ELK Biotechnology) following the manufacturer’s instructions. And the qRT-PCR for mRNAs and miRNAs was performed on a StepOne™ Real-Time PCR System (Life technologies) using EnTurbo™ SYBR Green PCR SuperMix (EQ001, ELK Biotechnology). The relative expression levels of mRNA or miRNA were normalized to those of GAPDH or U6 and evaluated using the 2^−ΔΔCT method. The primers used for qRT-PCR are listed in Table 1.

The expression of iNOS in ventricle was measured by real-time PCR. Total RNA was isolated from ventricular samples with Tripure Extraction Reagent (ELK Biotechnology, China) according to the manufacturer's protocol. cDNA was synthesized using EntiLink™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology, China). Real-time fluorescent quantitative PCR was performed on the StepOne real-time PCR instrument (Life technologies, Gaithersburg, MD), and each sample was made into three duplications using EnTurbo™ SYBR Green PCR SuperMix kit (ELK Biotechnology, China). Primers used for RT-PCR were: Dog-iNOS: 5'-ACCAATACAGGCTCGTGCAG-3'(forward),5'-GGGCTGTCTACTACTCGCTCC-3'(reverse);Dog-GAPDH:5'-GAAGGTCGGAGTGAACGGATT-3'(forward),5'-CATTTGATGTTGGCGGGATC-3'(reverse).