Sybr green qpcr mix kit

The mRNA expression levels of LC3, p62/SQSTM1, Beclin-1 and TFEB in aorta tissues and PPARα, PPARγ, SREBP1C and FAS in liver tissues were detected by quantitative real-time PCR. The primer sequences are given in Table 1. Brifly, total RNA from aorta and liver tissues was extracted by Trizol, and 1 μg of RNA was used to generate cDNA using a Primerscript RT Reagent Kit (Takara Bio). Reverse transcription reactions (RT) were performed according to the manufacturer’s instructions. The qRT-PCR analysis was performed in triplicate for target mRNAs using a qPCR SYBR Green Mix kit (Takara Bio), and the PCR conditions were as follows: 1 cycle of 95°C, 5 s; 40 cycles of 95°C, 10 s; 57°C, 30 s. The fluorescent signals of SYBR Green were subjected to cDNA analysis using an ABI 7900HT machine (Applied Biosystems, Forster, CA, USA). Melting curve analysis was performed to confirm specificity. The 2^-ΔΔCT method was used for the semi-quantitative PCR analysis. Target RNA levels were normalized to β-actin mRNA.

The MC3T3-E1 cells cultured on various nanofibrous matrices for 7 days were also collected for the evaluation of the osteogenesis-related genes expression. Total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. The total RNA concentration and purity were detected by a Nanodrop assay (Tecan M200), and the first strand cDNA was synthesized by reverse transcriptase as described in theM-MLV manual (Promega). The expression of osteogenic markers was quantified by qPCR SYBRGreen Mix Kit (TaKaRa). The primer sequences specific for the target gene including anti-runt-related transcription factor 2(RUNX2), osteopontin (OPN) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used for qRT-PCR are listed in Table 1. The specificities of the listed oligonucleotides were checked by BLASTN® (Basic Local Alignment Search Tool) against the mouse RefSeq RNA database at NCBI. The qPCR amplification was done as follows: initial denaturation at 95°C for 10min, followed by 40 cycles at 95°C for 30 s, 58°C for 1 min, 72°C for 1 min. The comparative threshold cycle method was used to analyse the Q-PCR results using iCycleriQ Detection System software with GAPDH as the reference gene. All results were quantified using the ΔΔCt relative quantification method.

Total RNA was isolated from the liver samples using the TRIzol agent (Invitrogen, Inc.) according to the supplier’s protocols. Next, purified RNA (1 μg) was used to generate first-strand cDNA with a Primescript RT Reagent Kit (Takara Bio) according to the manufacturer’s protocol. Real-time polymerase chain reaction (PCR) was performed using a qPCR SYBR Green Mix kit (Takara Bio) to analyze the mRNA expression levels of Nrf2, HO-1, TNF-α, IL1β, IL-6, farnesoid X receptor(FXR), PPARα, ACOX1, MCAD, CPT1, PPARγ, the sterol regulatory element binding protein-1C (SREBP-1c), fatty acid synthase (FAS) and SCD1. The primer sequences are listed in Table 1. Real-time quantitative PCR (qPCR) was performed on an ABI 7900HT machine (Applied Biosystems, Foster City, CA, USA). The relative expression level was calculated using the 2^-ΔΔCT method, and values were normalized to β-actin mRNA.