One step reverse transcription kit

Total RNA was extracted using TRIzol^® reagent according to the manufacturer's protocol (cat. no. 15596026; Thermo Fisher Scientific, Inc., Shanghai, China) and the concentration and purity of the RNA were determined using a Nanodrop 2000 spectrophotometer. RNA (2 µl) was used to synthesize cDNA using a one-step reverse transcription kit according to the manufacturer's protocol (Takara Biotechnology Co., Ltd., Dalian, China). Primers were synthesized by Shanghai Sangong Pharmaceutical Co., Ltd. (Shanghai, China). Primer sequences were as follows: Forward, 5′-TATTGAAACTCCTCGCCAGGA-3′ and reverse, 5′-GGCATAACGCATCTGGTTTT-3′ for BECN1; and forward, 5′-AGCCACATCGCTCAGACA-3′ and reverse, 5′-TCTCCTGGGAGGCATAGACC-3′ for GAPDH. Cycling conditions were as follows: An initial predenaturation step at 95°C for 5 min, followed by 37 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 13 sec and extension at 72°C for 30 sec. PCR products were detected by 2% agarose gel electrophoresis with ethidium bromide staining and the density of the bands was analyzed using ImageJ Software v1.48u (National Institutes of Health, Bethesda, MD, USA).

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to extract total RNA from rats. cDNA was synthesized from 1 µg of RNA using one-step reverse transcription kit (no. 639505; Takara Bio, Inc., Otsu, Japan). The mRNA levels of each index were measured using a fluorescence quantitative PCR kit (FSQ-101; Toyobo Life Science, Osaka, Japan). GAPDH was taken as an internal reference. TGF-β1 gene localization: NC_000019.10. Primers were designed and synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Sequence: upstream, 5′-GGCCAGATCCTGTCCAAGC-3′ and downstream, 5′-GTGGGTTTCCACCATTAGCAC-3′; internal reference GAPDH: upstream, 5′-TGGCCTTCCGTGTTCCTAC-3′ and downstream, 5′-GAGTTGCTGTTGAAGTCGCA-3′. The relative expression level of each index was calculated by 2^−ΔCq [ΔCq = Cq (target gene) - Cq (GAPDH)] (14 (link)–16 (link)).