Takara sybr premix ex taq

In order to study the expression of target genes in F. asiaticum, 200-μL aliquots of conidial suspensions (10⁶/mL) of test strains were inoculated on solid CM with cellophane overlays. After incubation at 23°C for 12 h, the samples were treated with either light or stress agents for 1 h. Subsequently, the mycelium samples were harvested followed by immediate freezing in liquid nitrogen. The frozen mycelium samples were subjected to RNA extraction by using Qiagen reagent (Germany). One microgram of each RNA sample was used for reverse transcription with the reverse transcription (RT) reagent kit (Perfect Real Time) (TaKaRa Biotechnology Co., Dalian, China) according to the manufacturer’s instructions. The resulting single-stranded cDNA was later used as a template for quantitative reverse transcription-PCR (qRT-PCR) in a CFX96TM real-time system (Bio-Rad, Inc., USA) using TaKaRa SYBR Premix Ex Taq (TaKaRa Biotechnology, Co., Dalian, China). Transcript levels of the target genes were normalized against tubulin gene expression. The experiment was repeated three times with triplicate samples. The gene expression levels were calculated using the 2^−ΔΔCT method. All primers used in the present study are listed in Table S1.

A total of 15 human gastric cancer tissues and paired adjacent non-tumor tissues (more than 2.5 cm from the edge of the cancer tissue), were surgically resected and first diagnosed for primary gastric cancer in the Foshan First People’s Hospital between January 2018 and December 2020. Patients who did not receive preoperative chemoradiation treatment were selected for this study. Ethical approval was obtained from the subject review committee of Foshan First People’s Hospital, and all 15 patients informed consent and signed the consent form. Gastric cancer tissues and adjacent tissues were immediately stored in liquid nitrogen after resection for further RNA extraction. Total tissue RNA was isolated using Trizol reagent (15596-026; Invitrogen; Thermo Fisher Scientific, Inc.), followed by cDNA synthesis using Prime-Script RT Master Mix (TaKaRa, Shiga, Japan) for reverse transcription and TaKaRa SYBR^® Premix Ex Taq™ (TaKaRa) for qRT-PCR. All primers used in the PCR are listed in Table 1. The expression levels of the four risk lncRNAs were normalized to the expression levels of GADPH.

Quantitative reverse-transcription PCR (RT-qPCR) was carried out to examine the relative abundance of target RNA. 0.1 μg RNA were used for cDNA synthesis using RevertAidTM First Strand cDNA Synthesis Kit (Thermo). Experiments were performed with Takara SYBR Premix Ex Taq (Takara) according to the manufacturer’s instructions and examined by a CFX96 Real-Time PCR System (Bio-Rad). *P < 0.05; **P < 0.01; ***P < 0.001. The primers used for RT-qPCR in this study are listed in Additional file 10.

Cell culture reagents, 5-bromo-2'-deoxyuridine (BrdU), anti-BrdU antibody, DNase I and TRIzol, were purchased from Life Technologies (Carlsbad, CA, USA). GLP-1, Exendin9-39 (Ex9), isobutylmethylxanthine (IBMX), dexamethasone (Dex), 4',6-diamidino-2-phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Oil Red O were acquired from Sigma Chemical (St. Louis, MO, USA). Complete Protease Inhibitor Cocktail was purchased from Roche Applied Science (Mannheim, Germany). TaKaRa PrimeScript^TM RT reagents kits and TaKaRa SYBR premix Ex Taq were acquired from TaKaRa Bio (Kyoto, Japan). Antibodies for β-catenin, (Ser³⁷/Thr⁴¹) non-phospho-β-catenin and FITC-conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Cell Signaling Transduction (Boston, MA, USA). Antibodies for Wnt4 and GAPDH were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lifectamine2000 and siRNA control were purchased from Life Technologies (Grand Island, New York, USA). All other chemicals of analytical grade were acquired from Dingguo Bio (Shanghai, China).