Sybr green qpcr master mix

The qRT-PCR was performed as described previously.18 (link) To determine the mRNA levels of STAT3 and USP28, qRT-PCR was performed using SYBR Green qPCR Master Mix (Clontech Laboratories, Inc., Palo Alto, CA, USA) with Roche LightCycler^® 480II real-time PCR system (Roche, Basel, Switzerland). The primers used were as follows: STAT3, forward 5′-CAGTGACCAGGCAGAAGA-3′ and reverse 5′-ACTCCATCGCTGACAAAA-3′; USP28, forward 5′-TGGGAAGGATTCTGGTTA-3′ and reverse 5′-GCTGATAGAGCCTGGAGTA-3′; GAPDH, forward 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse 5′-TGG TGAAGACGCCAGTGGA-3′.

RNA from cells or rat liver tissues was extracted by using TRIzol Reagent (Invitrogen, Waltham, MA, USA). RNA concentration and quality were detected by NanoDrop. cDNAs for mRNA expression detection were reverse transcribed with 5 μg total RNA per sample by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), and miRNA cDNAs were transcribed with 2 μg total RNA per sample by using the Mir-X miRNA qRT-PCR SYBR Kit (Clontech Laboratories, Mountain View, CA, USA). Quantitative real-time PCR was performed by using the SYBR Green qPCR Master Mix (Clontech, Mountain View, CA, USA) in the Agilent Mx3005P and Bio-Rad iQ5 systems. The detection program of gene expression with cDNA transcribed from mRNA is: 10 min at 95°C, 40 cycles of 10 s at 95°C, 10 s at annealing temperature, and 30 s at 72°C. The program of miRNA cDNA detection is: 10 min at 95°C, 40 cycles of 10 s at 95°C, and 30 s at 60°C. Melting curves were performed to check quantitative real-time PCR products. Data were normalized by β-actin for mRNA expression and by U6 for miRNAs. The gene primer information for quantitative real-time PCR is depicted in Table S3. miRNA primer information for quantitative real-time PCR is depicted in Table S4.

Base on the instruction of the tissue RNA extraction kit (Tiangen Biotechnology Co., Ltd., Beijing, China), total RNA was extracted. Then complementary DNA (cDNA) was synthesized using PrimeScript RT Reagent kit (TaKaRa, Dalian, China) with 1 μg RNA, and the qRT-PCR reaction was carried out by SYBR Green qPCR Master Mix (TaKaRa). The sequence of primers were as follows: TP73-AS1 forward, 5ʹ-GCTCCGTGAACCAACTCG-3ʹ, and reverse, 5ʹ-GCTGCCAAGGGAACTCT-3ʹ; miR-494-3p forward, 5ʹ- GGGTGAAACACACACGGGAA-3ʹ, and reverse, 5ʹ- GGCAGGTCCGAGGT-3ʹ; EIF-5A2 forward, 5ʹ-TTCCAGCACTTACCCTT-3ʹ, and reverse, 5ʹ-TTCCCCTCTATTTTTG-3ʹ; GAPDH forward, 5ʹ-TCGACAGTCAGCCGCATCTTCTTT-3ʹ, and reverse, 5ʹ-ACCAAATCCGTTGACTCCGACCTT-3ʹ; U6 forward, 5ʹ-CTCGCTTCGGCAGCACA-3ʹ, and reverse, 5′-AACGCTTCACGAATTTGCGT-3ʹ. GAPDH and U6 were used as inside parameters to normalize the data, and the mRNA expression level was calculated using 2^{−ΔΔ Ct}.