Sybr premix ex taq 2 kit

Total RNA was extracted and purified from colon and liver tissues using the Trizol tissue kit according to the manufacturer instructions (Tiangen, Beijing, China). Reverse transcription (RT) was performed using an RT Kit (Takara, Beijing, China). RT reaction mixtures contained 1 μg total RNA and 1× PrimeScript RT Master Mix in a final volume of 20 μL, and were conducted for 15 min at 37°C. Reverse transcriptase was inactivated by heating to 85°C for 5 s. Quantitative RT-PCR (qRT-PCR) assays were performed using SYBR Premix Ex Taq TM II Kit (Vazyme, Nanjing, China). The qRT-PCR reaction mixture contained 1 × SYBR Premix Ex Taq TM II, 0.4 μM each forward and reverse primers, and 2 μL cDNA templates in a final volume of 20 μL. Reactions were performed as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. The primers used are listed in Table 1 and were synthesized by Suzhou Genewiz Biological Co. (Genewiz, Suzhou, China). These mouse primers were selected from the Primer Bank developed by the Massachusetts General Hospital and Harvard Medical School (21 (link)). The relative expression levels of target genes were normalized to those of GAPDH and calculated using the 2^–ΔΔCT.

Total mRNA (the liver of chickens) was extracted using TRIzol (Vazyme, Nanjing, China). The total RNA purity and concentration were assessed according to the 260/280 nm absorbance ratio. The cDNA synthesis was performed with 1 µg total RNA using a PrimeScriptTMRT reagent kit (Vazyme, Nanjing, China).
Gene expression of β-actin, Nrf2, GSH-Px, GLRX2, Keap1, Bax, Caspase3, P53, AKT, PI3K and Bcl-2 was analyzed by qRT-PCR using the SYBR® Premix Ex TaqTM II kit (Vazyme, Nanjing, China) on an ABI Fluorescence Quantitative PCR Detection System (iQ5, ABI, Waltham, MA, USA). The analysis and primer pair synthesis (Table 1) were conducted by Sangon Biotech Co., Ltd. (Shanghai, China). All data were normalized to β-actin. Data were calculated by the 2^−ΔΔCt method.

To explore the impact of IjpacC deletion on the expression of acidic-expressed S53 genes, a S53 gene (IF1G_06234) was taken as a target, which was reported to upregulate significantly during the fungal infection in insects [16 (link)]. The wild-type and ΔIjpacC mutant strains were inoculated on the 3rd or 4th instar larvae of the potato tuberworm, respectively. After infection for 24, 48, and 72 h, larvae were collected. Each treatment had three repetitions. Fungal strains without infection were taken as controls (0 h). Total RNA was extracted from samples of each treatment larvae and cDNA was synthesized. Using cDNA as template, qRT-PCR was performed to detect expression levels of the target fragment of the fungal S53 gene with the primer pair IF1G_06234F/R, by using a SYBR Premix ExTaq^TM II kit (Vazyme, Nanjing, China) and a BIO-RAD CFX96 PCR system (BIO-RAD, Hercules, CA, USA). All reactions were run in triplicate. Expression of actin gene was taken as the internal control. The changes in relative gene expression in different time periods were calculated by the 2^−ΔΔCT method [31 (link)]. By comparing the relative expressions of the target gene (IF1G_06234F/R) in ΔIjpacC mutant and the wild-type strains, the impact of IjpacC deletion on S53 gene expression was observed.