Sybr premix ex taq reagent

Total RNA was isolated from the jejunal mucosa by using TRIZOL reagent (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) according to the manufacturer’s instructions. The RNA quality was determined by DU 640 UV spectrophotometer detection (Beckman Coulter Inc., Fullerton, CA), and the OD₂₆₀:OD₂₈₀ ratio ranged from 1.8 and 2.0 in all samples. The RNA integrity was analyzed by 1% agarose gel electrophoresis. The RNA samples were reversely transcribed into complementary DNA by using RT Reagents (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) according to the manufacturer’s instruction. Following reverse transcription, expression levels of ZO-1, occludin, Bcl-2, Bax and β-actin in the jejunal mucosa were analyzed by real-time quantitative PCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) and CFX-96 Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA) as previously described [32 (link)]. The primers were purchased by TaKaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China), which was listed in Table 2. Relative gene expression to the reference gene (β-actin) was determined in order to correct for the variance in amounts of RNA input in the reaction. In addition, the relative gene expressions compared to the reference gene were calculated with the previous method [33 (link)].

Metformin was purchased from Santa Cruz. Thiazolyl blue tetrazolium bromide was from Sigma-Aldrich. Culturex 96 well BME invasion assay kit was purchased from R&D systems. PrimeScript™ RT Master Mix and SYBR Premix Ex Taq reagents were purchased from Takara Biotechnology. The human AGS gastric cancer cell line was purchased from the National Cell Bank of Iran. DMEM/F12 medium and fetal bovine serum (FBS) were purchased from Gibco. TriPure isolation reagent was from Roche. Enhanced chemiluminescence reagent (ECL) was purchased from Najm Biotech. Antibodies against vimentin (sc-32322), β-catenin (sc-7963), E-cadherin (sc-7870) were purchased from Santa Cruz. GAPDH antibody (G9545) was from Sigma. HRP-linked anti-mouse (ab6789) and anti-rabbit (ab97051) IgG were purchased from Abcam.

Total RNA was extracted from the samples with TRIzol^® reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. The cDNA was prepared as described previously [48 (link)]. SYBR Premix Ex Taq reagents (TaKaRa, Dalian, China) were used for the qPCR assay. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Relative mRNA expression was calculated with the comparative cycle threshold (CT) (2^−ΔΔCT) method [49 (link)]. The qPCR primers used in this study are listed in Table 1.

Total RNA was extracted from primary cultured chondrocytes using the TRI reagent (Molecular Research Center, Inc.). For cartilage tissues, mouse knee joints were scraped with a blade after DMM surgery or sham operation, and RNA was isolated using the TRI reagent^{10 (link)–13 (link)}. Total RNA was reverse transcribed, and the resulting cDNA was PCR-amplified using the PCR primers and experimental conditions summarized in Supplementary Table 2. qRT-PCR was performed using an iCycler thermal cycler (Bio-Rad) and SYBR premixExTaq reagents (TaKaRa Bio). For each target gene, transcript levels were normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as fold-change relative to the indicated control.

Total RNAs were extracted using the TRIzol^® Reagent (Invitrogen) according to the manufacturer's protocol. Two-micrograms (μg) of RNAs were used as the template, and the M-MLV reverse transcriptase (Promega, Madison, WI, USA) and random hexamer primers (TaKaRa, Dalian, China) were used to synthesize cDNAs. The yield cDNAs were detected and quantified by quantitative real-time PCR (qPCR) using SYBR Premix Ex Taq reagents (TaKaRa) and the Mx3005P QPCR System (Agilent Technologies, Palo Alto, CA, USA). Relative abundance of mRNA was calculated using the comparative cycle threshold (CT) (2^−ΔΔCT) method.^{60 (link)} The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the endogenous control. All the qPCR experiments were performed three times. The data represent results from one of the triplicate experiments. *P<0.05 considered significant, **P<0.01 considered highly significant. The qPCR primers used in this study were shown in Table 1.