Sybr green real time pcr master mix kit

Total RNA was isolated from cells using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. One microgram of RNA was used for reverse transcription with gDNA Eraser reverse transcription kits (Toyobo). cDNA was used for quantification of the indicated mRNA on a QuantStudio 6 Flex RT-PCR System (Applied Biosystems) with SYBR Green Real-Time PCR Master Mix kits (Toyobo) in accordance with the manufacturer’s instructions. Dissociation curve analysis of the products was conducted at the end of each PCR cycle to detect and validate the specific amplification of PCR products. The transcript level of each gene was normalized to that of GAPDH, and the 2^−ΔΔCT method was used to analyze gene expression in the samples. Data are presented as fold changes compared to the corresponding level in untreated control cells. The primer sequences used for RT-qPCR are listed in Table 1.

RNA from lungs and spleens of mice was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). We performed cDNA synthesis using ReverTra Ace® qPCR RT kits (FSQ-101, Toyobo), and the relative mRNA expression of different genes was measured by quantitative real-time PCR with SYBR® Green Realtime PCR Master Mix kits (QPK-212, Toyobo) and calculated by comparison with the control gene Actb (encoding β-actin) using the 2^−△Ct method. The primers used are listed in Table 2.

To validate the RNA-seq data, nine genes were randomly chosen for validation using qRT-PCR. According to the manufacturer’s instructions, total RNA was extracted using a Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) from the samples of C. paliurus cuttings in the OC, IE, CF, and RT stages. A NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis were used to assess the total RNA concentration and quality. The cDNA was obtained using MonScript RTIII All-in-One Mix with dsDNase kits (Monad Biotech Co., Ltd., Suzhou, China). SYBR Green Realtime PCR Master Mix Kit (Toyobo Co., Ltd., Osaka, Japan) and an Applied BiosystemsTM 7500 Real-Time PCR System (Monad, China) were used for qRT-PCR analysis. Each sample included three biological and technical replicates. The National Center for Biotechnology Information (NCBI) online tool (http://www.ncbi.nlm.nih.gov/) was used to design the primers, and the primer sequences for qRT-PCR are shown in Table S7. The relative expression levels of the target genes were quantified using the 2^−ΔΔCt formula [59 (link)].

Total RNA was isolated from BCA samples and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, United States). Sample RNA (1 μg) was reverse transcribed using a Reverse Transcription Kit (Takara, Dalian, China). Quantitative real-time PCR was performed using an SYBR Green Real-time PCR Master Mix kit (Toyobo, Osaka, Japan) on the 7900HT fast real-time PCR system (Applied Biosystems, San Francisco, CA, United States). The levels of miR-99b were quantified with the mirVana^TM qRT-PCR microRNA Detection Kit (Ambion, Austin, TX, United States). The primers used in this study were synthesized by Genepharma (Shanghai, China). The primer sequences for qRT-PCR were purchase from Genepharma (Shanghai, China). The results were normalized to the levels of GAPDH or U6, respectively.

The mRNA levels of nitric oxide synthase 2 (NOS2), arginase 1 (ARG1), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interleukin-6 (IL6), interleukin-10 (IL-10), chemokine (C-X-C motif) ligand 2 (CXCL2), cluster of differentiation 86 (CD86) and cluster of differentiation 206 (CD206) were detected by real-time quantitative PCR (RT-qPCR). Firstly, RAW264.7 cells were exposed to 0 or 50 μM AFB1 for 24 h. After treatment, cells were washed with PBS and harvested into 1 mL TRIzol (Invitrogen, Waltham, MA, USA) and total RNAs were extracted according to the manufacturer’s instructions. The quantities and qualities of RNAs were evaluated using a NanoPhotometer® N60/N50 (Implen, München, Germany). The complementary DNA (cDNA) was reverse-transcribed from 10 ng of total RNA using a ReverTra Ace^® qPCR RT Master Mix with gDNA Remover Kit (TOYOBO, Osaka, Japan). The levels of the transcripts of the target genes were determined using SYBR^® Green Realtime PCR Master Mix Kit (TOYOBO, Osaka, Japan) on an RT-qPCR system (BIO-RAD, Hercules, CA, USA). The relative amount of target mRNA was performed using the −2^−ΔΔCt method with GAPDH as a reference gene. The primers used in RT-qPCR reactions were synthesized by Sangon Biotech (Shanghai, China) and are showed in Table 3.