One step iscript cdna synthesis kit

RT‐qPCR was conducted to analyse gene expression in fungal strains. Fungal RNA was purified using TRIzol reagent (Sigma‐Aldrich), treated with RQ1 RNase‐free DNase (Promega), and used for the synthesis of complementary DNA with a One‐Step iScript cDNA synthesis kit (Bio‐Rad). Reactions for qPCR were prepared using iQ SYBR Green Supermix (Bio‐Rad) and carried out in a CFX Connect Real‐Time PCR Detection System (Bio‐Rad). All primers are listed in Table S1. The β‐tubulin‐coding gene was used as an internal control. The 2^−ΔΔCt method was used to determine relative expression levels of genes. All treatments were performed with three technical replicates, and significant differences were determined by statistical analysis. Experiments were repeated at least two times.

Cells were lysed, and total RNA was extracted using RNeasy extraction kit (Invitrogen). cDNA was generated by one-step iScript cDNA Synthesis Kit (Bio-Rad), and qRT-PCR was performed using the EvaGreen qPCR MasterMix (abm) and CFX96 Real-Time PCR System (Bio-Rad). Relative quantification was expressed as 2^−ΔCt, where C_t is the difference between the main C_t value of triplicate of the sample and that of an endogenous L19 or GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA control. The human and mouse primer sequences used are listed in the Supplementary Materials.

Cells were lysed, and total RNA was extracted using the RNAeasy extraction kit (Invitrogen). cDNA was generated using the one-step iScript cDNA synthesis kit (Bio-Rad), and quantitative real-time PCR was performed using the EvaGreen qPCR MasterMix (Abm) and CFX96 real-time PCR system (Bio-Rad). Relative quantification was expressed as 2^−Ct, where −Ct is the difference between the main Ct value of triplicates of the sample and that of an endogenous L19 or GAPDH mRNA control. The mouse or VSV primer sequences used are listed in the Supplemental Material.