Sybr green

Total RNA was extracted from aortas and human VSMCs using RNApure total RNA fast isolation kits (BioTeke, Beijing, China) according to the manufacturer’s protocols. The concentration of total RNA was quantified by a spectrophotometer (Thermo Fisher Scientific). Reverse transcription was performed to synthesize cDNA using reverse transcriptase M-MLV (Takara, Otsu, Japan) and oligo (dT)15 and random primers or specific miRNA RT primers. The quantitative real-time polymerase chain reaction (qRT-PCR) of miR-377-3p and NRP2 was performed with SYBR Green (BioTeke) and Taq HS Perfect Mix (Takara). U19 and β-actin were used to normalize miR-377-3p and NRP2 expression, respectively. The relative levels were presented as 2^−ΔΔC_t. The primer sequences used in the present study were listed in Table 1.

Total RNA was extracted from ovary or skeletal muscle tissues using a RNApure total RNA isolation kit (Bio Teke, Beijing, China). The concentration of RNA was detected on an ultraviolet spectrophotometer (Nana Drop 2000, Thermo Scientific, USA). Subsequently, cDNA was synthesized using M-MLV Reverse Transcriptase (Takara, Japan). Real time PCR was carried out using Taq HS Perfect Mix (Takara) and SYBR Green (Bio Teke) on an Exicycler™ 96 Real-Time Quantitative Thermal Block ⁽BIONEER, Korea). The primer sequences are presented in Table 1. The mRNA expression level was calculated by 2^-△△CT method.
Table 1

Oligonucleotide primer sets for real-time PCR

Name	Sequence (5′¬3′)	Length
CYP17 F	TGGAGGTGATAAAGGGTT	18
CYP17 R	CGTCAGGCTGGAGATAGA	18
CYP19 F	GCCTGTCGTGGACTTGGT	18
CYP19 R	TAAATTCATTGGGCTTGG	18
PPARγ F	TACCACGGTTGATTTCTC	18
PPARγ R	AATAATAAGGCGGGGACG	18
PGC-1α F	GAACAAGACTATTGAGCGAACC	22
PGC-1α R	GAGTGGCTGCCTTGGGTA	18
β-actin F	CCACTGCCGCATCCTCTT	18
β-actin R	GGTCTTTACGGATGTCAACG	20

The total RNA was extracted with RNApure extraction kit (BioTeke, Beijing, China), and the concentration was measured with NANO 2000 ultraviolet spectrophotometer (Thermo Scientific, Waltham, MA, USA). The RNA was reversely transcribed into cDNA with M-MLV reverse transcription (TAKARA, Japan) with Oligo(dT) and random primer. The instruments and reagents used in reverse transcription were RNase-free. The cDNA was used for real-time PCR with Taq HS Perfect Mix (TAKARA) and SYBR Green (BioTeke) to detect the mRNA levels of ANGPTL8, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. β-actin served as the internal control. The PCR procedure was set as follow: 94°C for 5 min 20 s, 60°C for 30 s, 72°C for 40 s, and 40 cycles of 72°C for 5 min 30 s, 40°C for 4 min 30 s, melting 60-90°C every 1°C for 1 s, and incubated at 25°C for several minutes. The PCR ran in Exicycler TM96 quantitative thermal block. The data were calculated using 2^-ΔΔCt method. The primers were purchased from Genscript (Nanjing, Jiangsu, China), and the information was shown in Table 1.

Total RNAs were isolated from SMCs through TRIpure reagent (BioTeke, Beijing, China), and then reversely transcribed into cDNA using Reverse Transcriptase M‐MLV (Takara, Dalian, China) in the presence of random hexamers and oligo (dT). RT‐qPCR was performed by using SYBR Green (BioTeke) and the primer sequences were listed in Table S1. The 2^−ΔΔct method was used to determine relative gene expression.

The messenger ribonucleic acid (mRNA) expression levels were measured via RT-qPCR using the 2× Power Taq PCR Master Mix (BioTeke, Beijing, China) and SYBR Green (BioTeke, Beijing, China) and an ExicyclerTM 96 fluorescence quantitative analyzer. Total RNAs were extracted from myocardial tissues using TRIzol (Invitrogen) and reversely transcribed and synthesized into complementary deoxyribonucleic acids (cDNAs). Then, the concentration and purity of RNAs were detected using Thermo NanoDrop 2000. The synthesis of cDNAs was conducted using 1 μg of total RNAs, reverse transcriptase (Fermentas), and Oligo-dT primers. In PCR amplification, a 50 μL of PCR system was utilized, which contained a reaction buffer, Taq DNA polymerase, dNTPs, 1 μL of forward primer, 1 μL of reverse primer, and 3 μL of template cDNAs (resulting products of reverse transcription reaction). The samples were loaded, shaken for blending, and transiently centrifuged, followed by amplification using a PCR instrument. The recommended reaction conditions are as follows: denaturation at 94°C for 20 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, and extension at 72°C for 30 s. PCR was performed using an 7900 QPCR system (Applied Biosystems), with the following cycle parameters: 95°C for 5 min, 1 cycle, and 95°C for 10 s and 60°C for 20 s, 40 cycles. The sequences are shown in Table 1.