Sybr green pcr kit

Total RNA from the liver was extracted using Trizol reagent (Cat# R0016, Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer’s protocol and reverse transcribed into cDNA using a cDNA synthesis kit (Cat# AG11728, Accurate Biotechnology Co., Ltd., Hunan, China). The gene expressions of SIRT1, PGC-1α, Nrf1, Nrf2, TNF-α, and IL-1β were detected using an ABI QuantStudio™ 6 system (Applied Biosystems; Thermo Fisher Scientific, Inc. (Waltham, MA, USA)) with the SYBR Green PCR kit (Cat# AG11718, Accurate Biotechnology Co., Ltd., Changsha, China). The primers were synthesized by Shanghai Sangon Biological Engineering Co., Ltd. (Shanghai, China) Relative quantification was calculated using the 2^−∆∆Ct method. Primer sequences are shown in Table 1.

Another half of the samples mentioned above were cut into pieces, and ground with small steel balls in a cold mill. Total RNA was isolated from the samples by using Trizol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription from total RNA (1000 ng) was performed using Evo M-MLV RT Mix Kit (AG11728, Accurate Biotechnology, Hunan, Co., Ltd) with gDNA Clean for qPCR. RT-PCR reactions were carried out using the SYBR Green PCR Kit (AG11701, Accurate Biotechnology, Hunan, Co., Ltd). After a 30-s predegeneration at 95°C, 40 cycles were performed: denaturation at 95 seconds for 15 seconds, extension at 60°C for 30 seconds followed by a 65 to 95°C solubility curve, which was constructed to analyze the fluorescence signal. The relative amount of mRNA was normalized against GAPDH (Supplementary Information 1). The primers (Table 1) were produced by Shanghai Sangong Biotechnology (Shanghai, China). The relative expression levels were determined by the 2^−ΔΔCT method and calculated relative to the control group.

Total RNA was extracted from PDAC cells, tissues and matched non-cancerous tissues with TRIzol Reagent (Invitrogen, USA) in line with the manufacturer’s instructions. gDNA was isolated using a gDNA Reagent Kit (Sangon Biotech, Shanghai, China). cDNA was synthesized from 1 μg of total RNA using a PrimeScript RT Reagent Kit (Accurate Biology, China). qRT-PCR was performed with a SYBR Green PCR Kit (Accurate Biology, China) in an ABI 7900HT System (Applied Biosystems, USA) to determine the expression levels of candidate genes. The relative circRNA/mRNA and miRNA expression levels were normalized to those of GAPDH and U6, respectively, by using the 2 ^−△△CT method. For RNase R treatment, 10 μg of total RNA from the indicated cells was mixed with or without RNase R (3 U/μg) for 20 min at 37 °C. Next, circEYA3 and linear EYA3 mRNA were reverse transcribed with specific primers and analysed by qRT-PCR or PCR followed by nucleic acid agarose gel electrophoresis. For ActD treatment, cells were exposed to 2 μg/mL ActD (Sigma) at the indicated times. Then, the expression of the circRNA or linear mRNA was detected and analysed using qRT-PCR. Each experiment was carried out at least three times. All primers used in the experiment were synthesized by NovaBio (Shanghai, China) and are listed Additional file 1:Table S1.