Superscript 3 rt

Total RNA was extracted from cells using TRIzol reagent (TaKaRa) according to the instructions. After measuring the RNA concentration with a Nanodrop 2000, reverse transcription was performed using Superscript III RT (TaKaRa). Quantitative real-time PCR was performed on an Applied Biosystem StepOne Plus PCR system (ABI) using SYBR Green PCR Master Mix (TaKaRa). All primers were designed based on NCBI reference sequences and synthesized by Invitrogen (Suzhou, China). Primer sequences were listed in Supplemental Table S1. Target gene expression was calculated using the 2^−ΔΔCt method. Each experiment was performed in triplicate at least three times.

RAW264.7 cells were seeded in a 24-well microplate (1x10⁶ cells/ml) and were cultured for 24 h in the presence of SHP (100 µg/ml) or LPS (1 µg/ml); cells treated with RPMI were used as the negative control group and LPS as the positive control group. Total RNA was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA generation was carried out in a total volume of 10 µl, containing 5 µl RNA (100 ng/ml), 0.5 µl 10 pmol oligo-(dT)20 primer, 0.5 µl dNTP (10 mM) and 4 µl superscript III RT (Takara Biotechnology, Co., Ltd.). The primers used are shown in Table I, and β-actin was used as the internal standard. Quantitative PCR (qPCR) was performed using the CFX connect Real-time system (Bio-Rad Laboratories, Inc.) with a Fast Start DNA Master TB Green II kit (Takara Biotechnology Co., Ltd.), with the following program: 1 cycle of initial PCR denaturation at 95˚C for 15 min, 2 cycles of primer annealing at 60˚C for 0.5 min, 32 cycles of denaturation at 95˚C for 0.5 min, 1 cycle of final extension at 60˚C for 1 min and 1 cycle of melting curve analysis at 95˚C for 0.5 min. The results were calculated using the 2^-∆∆Cq method (20 (link)) and are expressed relative to β-actin.