Two step rt pcr supermix

Eight DEGs were selected for RT-qPCR evaluation to evaluate the accuracy of the transcriptome data obtained by Illumina sequencing. The cDNA at the two life stages was reversely transcribed using One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Then, qPCR was performed using Two-Step RT-PCR SuperMix (TransGen Biotech) and a Takara qPCR instrument. With Mj-actin as a reference gene, the relative fold change was calculated using the 2^−ΔΔCt method [31 (link)]. Each experiment was conducted in triplicate, with three biological replicates each.

An RNA prep micro kit (Tiangen, Beijing, China) was utilized to extract the RNA from M. javanica at different developmental time points, including Pre-J2, 2 dpi and 5 dpi (Par-J2), parasitic third-stage juveniles at 10 dpi and 14 dpi (Par-J3), parasitic fourth-stage juveniles at 18 dpi (Par-J4), and adult female nematodes at 30 dpi. Approximately 100 nematodes at Pre-J2, Par-J3, Par-J4 and female stages were collected for RNA extraction. Regarding the Par-J2, 100 nematodes were inoculated into the tomato root, and the infected roots were collected for RNA extraction. Next, the cDNA at seven life stages was subjected to reverse transcription using One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech), followed by qPCR using Two-Step RT-PCR SuperMix (TransGen Biotech) and a Takara qPCR instrument. With Mj-actin as a reference gene, the relative transcript abundance was calculated using the 2^−ΔΔCt method [29 (link)]. Each experiment was conducted in triplicate, with three biological replicates each.

Cell Counting Kit-8 (CCK-8), NADP + /NADPH assay kit, Bicinchoninic acid (BCA) protein assay kit, radioimmunoprecipitation assay (RIPA) lysis buffer, Mito-Tracker Green staining solution and Hoechst 33342 live cell staining solution were purchased from Beyotime (Shanghai, China). ELISA assay kit was purchased from mlbio (Shanghai, China). 4’, 6-Diamidine-2’-phenylindole dihydrochloride (DAPI), coverslips and Triton X-100, penicillin, streptomycin and bovine serum albumin (BSA) was purchased from Solarbio (Beijing, China). Enhanced chemiluminescence (ECL) kits were obtained from Thermo Fisher Scientific Pierce (Rockford, IL, USA). Fetal Bovine Serum (FBS) and Dulbecco’s Modified Eagle’s medium (DMEM) were purchased from Hyclone (Logan, UT, USA). Rotenone was purchased from MCE (Shanghai, China). Mito-SOX red mitochondrial superoxide indicator was purchased from Yeasen (Shanghai, China). Trizol Reagent, cDNA synthesis superMix and Two-step RT-PCR superMix were purchased from TransGen Biotech (Beijing, China). Primary antibodies, including NLRP3, ASC, Caspase-1, IL-1β and α-Tubulin, were purchased from Proteintech (Wuhan, China), and NF-κB p65, Phospho-NF-κB p65, IκBα and Phospho-IκBα were purchased from Cell Signaling Technology (Danvers, MA, USA). Peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Proteintech (Wuhan, China).