Sybr green 1

Total RNA was extracted from AT and serum and quantified (300 ng/μL RNA of each sample), respectively. Reverse transcription was performed using the FSQ-301 kit (TOYOBO, Shanghai, China) according to the manufacturer’s instructions. QRT-PCR system for mRNAs was 10 μL, containing 0.6 μL cDNA, 0.3 μL of each primer, 5 μL 2 × SYBR Green I (TOYOBO, Shanghai, China), 0.2 μL 50 × ROX (TOYOBO, Shanghai, China), 3.6 μL RNA-free water. QRT-PCR system for miRNA was 10 μL, including 0.8 μL cDNA, 0.3 μL of each primer, 5 μL 2 × SYBR Green I (TOYOBO, Shanghai, China) 0.2 μL 50 × ROX (TOYOBO, Shanghai, China), 3.4 μL RNA-free water. QRT-PCR procedures of the mRNAs and miRNAs were: 95 °C for 1 min, followed by 45 cycles, 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, and 72 °C final extension for 30 s. The β-actin gene and U6 were selected as the internal references mRNAs and miRNAs, respectively. All primer sequences are provided in Table 1.

Total RNA was extracted from different tissues using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Agarose gel electrophoresis (AGE) was used to estimate the integrity of total RNA, and a DU800 spectrophotometer (Beckman Coulter, Miami, FL, USA) was used to quantify the total RNA. Briefly, 300 ng of RNA of each sample was reverse-transcribed using the reverse transcription kit FSQ-301 (TOYOBO, Shanghai, China) following the manufacturer’s instructions. qRT-PCR amplification efficiency was assessed by varying the cDNA template concentration to obtain a relative quantitative standard curve, and specificity was assessed by melting curve. qRT-PCR was performed in three technical iterations with a 10 μL reaction mixture, which contained 5 μL of 2× SYBR Green I (TOYOBO, Shanghai, China), 0.2 μL of 50× ROX reference dye (TOYOBO, Shanghai, China), 0.3 μL of each primer, 1 μL of cDNA, and 3.2 μL of RNA-free water. U6 was chosen as an endogenous control for miRNAs. The qRT-PCR procedure was as follows: 95 °C for 1 min, followed by 40 cycles at 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, and s final extension for 30 s at 72 °C. All primer sequences are provided in Table 1.

Quantitative real-time PCR (qRT-PCR) was used to analyze the transcriptional activities of Arih2 gene in each tissue. The qRT-PCR system doses and reaction procedures of the Arih2 gene were the same as the internal reference β-actin (GenBank number: NM205518). The primers used for qRT-PCR were as follows: target gene Arih2: 5′-AGGGACTATGTGGAGAGCCATTACC-3′ and 5′-AAGCAGAAGACCTCATTGCAACGAT-3′; internal reference gene β-actin: 5′-CACCAACTGGGATGATAT-3′ and 5′-CGTACTCCTGCTTGCTGATC-3′. qRT-PCR total system was 20 μl including: 10 μl 1× SYBR Green I (TOYOBO, Shanghai), 0.4 μl 50× ROX reference dye (TOYOBO, Shanghai), 0.6 μl of each primer, 2 μl cDNA and 6.4 μl ddH₂O. The reaction procedure was as follows: 95°C/1 min; 40 cycles of 95°C/15 s, 56°C/30 s, 72°C/40 s.