Trizol reagent

The total RNA of adipose tissue was extracted using the Trizol Reagent (InvivoGen, San Diego, CA, USA). After identifying the RNA integrity by electrophoresis, complementary DNA (cDNA) was synthesized by reverse transcription of 2 μg of total RNA. The primer reaction system: 10× PCR 2.5 μL, MgCl₂ (25 mmol/L) 2.5 μL, deoxy-ribonucleoside triphosphate (10 mmol/L), 0.5 μL, Taq polymerase (5 U/μL) 0.25 μL, VDR (PPARγ) forward primer (F) (100 μmol/L) 0.04 μL, VDR (PPARγ) reverse primer (R) (100 μmol/L) 0.04 μL, VDR (PPARγ) probe (P) (100 μmol/L) 0.04 μL; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F) (100 μmol/L) 0.04 μL, GAPDH (R) (100 μmol/L) 0.04 μL, GAPDH (P) (100 μmol/L) 0.04 μL, sterilized double distilled water supplement 17.01 μL, cDNA 2 μL, a total reaction volume of 25 μL. The amplification conditions of the PCR instrument (Roche 480II) were: pre-denaturation at 95°C for 3 min; 95°C for 5 s, 60°C for 15 s (temperature conversion rate is 20°C/s), and amplification rounds of 40 cycles at 40°C for 1 min per cycle. The fluorescence signals were acquired during the period of 60°C extension. The results were expressed as the ratio of the relative expression of the gene of interest to the expression of the internal reference gene GAPDH. Primer probe sequences in reaction system of real-time PCR are shown in Table 1.

For extracting total RNA TRIzol reagent was used (Invivogen, USA). The isolated RNA was submitted for reverse transcription using RT Reverse Transcription Kit (ThermoFisher USA). The cDNA was submitted for qRT-PCR using ABI Prism 7000 sequence detecting system (Biocompare, USA) with the help of SYBR^® Green qPCR supermix (Bio-Rad, USA).The fold changes in the expression were evaluated by comparative threshold cycle (Ct) method. GAPDH and U6 were used as controls for mRNA and miR respectively.