Stepone real time pcr instrument

TRIzol^® reagent (Thermo Fisher Scientific) was used to isolate total RNA from chondrocytes. To synthesize first-strand cDNA, an EntiLink^™ 1st Strand cDNA Synthesis Kit (ELK Biotechnology) was used. Then, RT-qPCR was performed on a StepOne™ real-time PCR instrument (Life Technologies) with EnTurbo^™ SYBR Green PCR SuperMix Kit (ELK Biotechnology Co., Wuhan, China). Primer sequences were: β-actin, Forward: 5’-GTCCACCGCAAATGCTTCTA-3’, Reverse: 5’-TGCTGTCACCTTCACCGTTC-3’; hsa-circ-0134111, Forward: 5’-GAAAACAGATGAGGAGAAGGCC-3’, Reverse: 5’-CGTCTTTTTCTCAGCTTTGCC-3’; CCL1, Forward: 5’-CCAGATGTTGCTTCTCATTTGC-3’, Reverse: 5’-CAGGGCAGAAGGAATGGTGT-3’; U6, Forward: 5’-CTCGCTTCGGCAGCACAT-3’, Reverse: 5’-AACGCTTCACGAATTTGCGT-3’; hsa-miR-224-5p, Forward: 5’-CAAGTCACTAGTGGTTCCGTTTAG-3’, Reverse: 5’-CTCAACTGGTGTCGTGGAGTC-3’; miR-515-5p Forward: 5’-CGGGTTCTCCAAAAGAAAGCA-3’, Reverse: 5’-CAGCCACAAAAGAGCACAAT-3’. U6 was used as the internal control for hsa-miR-224-5p and miR-515-5p. The internal reference for the other genes was β-actin, and we used the 2^–ΔΔCT method for analysis of gene expression.

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).

The total RNA of rat lung tissues was extracted using TRIzol reagent according to the manufacturer's instructions (Takara, Japan). A real-time quantitative reverse transcription-PCR (RT-PCR) analysis was performed by using a SYBR Premix Ex Taq™ Kit (Takara, Japan), and the reactions were conducted on a StepOne™ Real-Time PCR instrument (Life Technologies, Grand Island, NY). The primers used for PCR were as follows: caspase-3 forward 5′-actactgccggagtctgact-3′; reverse 5′-taaccgggtgcggtagagta-3′; Bax forward 5′-gaaccatcatgggctggaca-3′; reverse 5′-gtgagtgaggcagtgaggac-3′; Bcl-2 forward 5′-cttctctcgtcgctaccgtc-3′; reverse 5′-ggggtgacatctccctgttg-3′; Akt forward 5′-gagaaccgtgtcctgcagaa-3′; reverse 5′-gttctccagcttgaggtccc-3′; and GAPDH forward 5′-tgatgggtgtgaaccacgag-3′; reverse 5′-agtgatggcatggactgtgg-3′. PCR conditions were as follows: 95°C for 5 min; 35 cycles at 95°C for 20s, 60°C for 20s, and 72°C for 45 s. GAPDH was selected as an internal control, and the target gene expression was normalized to GAPDH expression and calculated using the 2^−ΔΔCt method.

The real‐time fluorescent quantitative PCR (RT‐PCR) was performed according to a previously reported protocol 17. Briefly, the total RNA was extracted on days 3, 7 and 14 of treatment with or without NPWT using TRIzol reagent (Invitrogen). The first strand of cDNA was obtained from the total RNA using oligo‐dT primers and reverse transcriptase (Takara Bio, Kusatsu, Shiga, Japan). RT‐PCR was completed in the StepOne Real‐Time PCR instrument (Life Technologies, Waltham, MA, USA). GAPDH mRNA expression was used as an endogenous control. The fold changes were calculated according to the manufacturer's instructions (Takara Bio). The PCR contained a first step of denaturation at 95°C for 1 min., followed by total 40 cycles at 95°C for 15 sec., 58°C for 20 sec. and 72°C for 45 sec., followed by measurement using the 2−ΔΔCt method. The primer sequences used in the present study are listed in Table. 1.

Total RNA was extracted from the isolated exosomes and atrial tissues using TRIpure Total RNA Extraction Reagent (ELK Biotechnology, China) according to the protocols of the manufacturers. The relative levels of miR-21-5p and U6 were detected by Stem-Loop RT-qPCR using an Enturbo™ SYBR Green PCR Supermix Kit (ELK Biotechnology, China) with a StepOne™ Real-Time PCR instrument (Life Technology, USA). The data were analyzed by 2^−ΔΔCT method.

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).