Sybr green qpcr master mix kit

Total RNA was extracted from liver tissues using a commercial reagent (Invitrogen, CA, USA) according to the manufacture’s protocol. The cDNA synthesis was performed by reverse transcription of 1 µg total RNA using Frist Strand cDNA Synthesis Kit (Thermo, USA). RT-PCR amplification was carried out with an SYBR Green qPCR Master Mix kit (Thermo, USA) according to the manufacturer’s protocol. The qPCR was carried out in duplicate, the condition of RT-PCR amplification reaction as follows: 45 cycles of 95 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s with the primer sequences (Table 1). The expression of target gene transcripts was related to the reference gene (GAPDH). Results were expressed as folds of control.Table 1

Sequences of primers used quantitative real-time PCR

Gene	Forward primer	Reverse primer
SCD-1	5′-TGCTGATCCCCACAATTCCC-3′	5′-CTTTGACGGCTGGGTGTTTG-3′
SREBP-1C	5′-CCCTGCGAAGTGCTCACAA-3′	5′-GCGTTTCTACCACTTCAGGTTTCA-3′
ACC	5′-ACACTGGCTGGCTGGACAG-3′	5′-CACACAACTCCCAACATGGTG-3′
FAS	5′-GGCCACCTCAGTCCTGTTAT-3′	5′-AGGGTCCAGCTGAGGGTACA-3′
GAPDH	5′-GAACGGGAAGCTCACTGGC-3′	5′-GCATGTCAGATCCACAACGG-3’

Total RNA was extracted from 3 × 10⁶ CD4⁺ T cells using the RNAeasy Mini Kit (Qiagen), following the manufacturer's instructions. cDNA was synthesized from 1 μg of RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Hanover, MD, USA). The cDNA obtained was diluted 1 : 4 and used in quantitative RT-PCR reactions using SYBR Green (qPCR Master Mix kit, Thermo Scientific). The following primers were used: FOXP3, Fwd: 5′-ACC TTC CCA AAT CCC AGT GC-3′ and Rv: 5′-CCT GGC AGT GCT TGA GGA AGT-3′; TGF-β, Fwd: 5′ CAG CAA CAA TTC CTG GCG ATA-3′ and Rv: 5′-AAG GCG AAA GCC CTC AAT TT-3′; IL-10, Fwd: 5′-GCT GAG AAC CAA GAC CCA GAC-3′ and Rv: 5′-GGA AGA AAT CGA TGA CAG CG-3′; indoleamine 2, 3-dioxygenase (IDO), Fwd: 5′-ACA GAA TGC TGG TGG AGG AC-3′ and Rv: 5′ GGA AGT TCC TGT GAG CTG GT-3′; and ubiquitin (UBQ) Fwd: 5′-CAC TTG GTC CTG CGC TTG A-3′ and Rv: 5′-CAA TTG GGA ATG CAA CAA CTT TAT-3′. The CFX96 Real-Time PCR Detection System (Biorad, Hercules, CA, USA) was used to obtain cycle threshold (Ct) values and the expression levels of target genes were normalized relative to the expression of UBQ as reference gene, using the equation 1.8−[^ΔC], where 1.8 correspond to mean PCR efficiency of 80%. The data are expressed as relative units (RU).

Macrophage colony-stimulating factor enzyme-linked immunosorbent assay (ELISA) kit (YM-QP10199; Shanghai YuanMu Biological Technology Co., Ltd., Shanghai, China), a multifunctional ELISA microplate (Infinite 200 PRO; Coslan scientific LTD, Guangzhou, China), Light Cycler real-time fluorescence quantitative PCR instrument (Roche, Basel, Switzerland), total RNA extraction kit (Solarbio R1200; Shanghai Hengfei Refrigeration Engineering Equipment Co., Ltd., Shanghai, China), M-MLV reverse transcription kit (Vazyme, Nanjing, China), ultraviolet spectrophotometer (Multiskan Sky; Thermo Fisher, Shanghai, China), qReal-time PCR kit (Invitrogen, Grand Island, New York), and SYBR Green qPCR Master Mix kit (Thermo Fisher) were obtained. The primers for miR-21, miR-124, and the internal reference (U6) were synthesized by Sangon Biotech Co, Ltd. (Shanghai, China, Table 1).

The mRNA expression of OPG, RANKL, runt-related transcription factor 2 (RunX2), and OCN in tissue from the right femur was assayed by RT-qPCR. Briefly, total RNA was isolated from frozen femur tissue using Trizol reagent (Takara, China) according to the manufacturer's protocol. The cDNA was synthesized from total RNA using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific, USA). RT-PCR was performed with a SYBR Green qPCR Master Mix kit (Thermo Fisher Scientific). The qPCR was performed in duplicate, the condition of RT-PCR amplification reaction was as follows: 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 15 s with the primer sequences (Table 1); GAPDH acted as an internal control. The relative mRNA expression was calculated by the 2^−ΔΔCT method.
Table 1.

Genes	Forward primer	Reverse primer
OPG	5′-TACAGCATCACTACGTAGGAC-3′	5′-ACGTCATGCGATCACATATCG-3′
RANKL	5′-GACAGGCACGGACTCGTA-3′	5′-CGCTCATGCTAGTCGTCTA-3′
Runx2	5′-AGCGCTTCTCAGGAGTTCCA-3′	5′-GCCGGGCCACATCGA-3′
OCN	5′-CTGGCTGCGCTCTGTCTCT-3′	5′-TGCTTGGACATGAAGGCTTTG-3′
GAPDH	5′-CAACTTTGGCATTGTGGAAGG-3′	5′-ACACATTGGGGGTAGGAACAC-3′

Total RNA was extracted from frozen rat liver tissues using Trizol Reagent
(Invitrogen, USA), following the manufacturer's protocol. cDNA synthesis was
carried out using the First Strand cDNA Synthesis kit (Thermo Fisher
Scientific). Real-time PCR was carried out using a SYBR Green qPCR Master Mix
kit (Thermo Fisher Scientific). The qPCR was performed in duplicate, the
parameter of RT-PCR amplification reaction was as follows: 40 cycles of 95°C for
10 s, 60°C for 15 s, and 72°C for 30 s, with the primer sequences shown in Table 2. Target mRNA expression of each
sample was normalized to endogenous control gene GAPDH.
Table 2.

Gene	Forward primer	Reverse primer
ACC	5′-ACACTGGCTGGCTGGACAG-3′	5′-CACACAACTCCCAACATGGTG-3′
SREBP-1C	5′-CCCTGCGAAGTGCTCACAA-3′	5′-GCGTTTCTACCACTTCAGGTTTCA-3′
LDLR	5′-CCAACCTGAAGAATGTGGTG-3′	5′-CAGGTCCTCACTGATGATGG-3′
PPARα	5′-GGAAACTGCCGACCTCAAAT-3′	5′-AACGAAGGGCGGGTTATTG-3′
GAPDH	5′-GAACGGGAAGCTCACTGGC-3′	5′-GCATGTCAGATCCACAACGG-3′

RNA was extracted with a RNeasy® Mini kit (Qiagen GmbH, Hilden, Germany), and converted into cDNA using a Revert Aid First stand cDNA synthesis Kit (Thermo Scientific, United States). Real-time PCR was performed using a SYBR Green/qPCR Master Mix kit according to the manufacturer’s instructions (Thermo Scientific, United States). Real-time RT-PCR was performed in triplicate and normalized to GAPDH or β-actin with the ΔΔCt method. Primers are listed in Table 1.