Fastking reverse transcriptase kit

Total RNA of the kidney was extracted using Trizol reagent (Tiangen, China) according to the manufacturer's protocol. The concentration and purity of isolated RNA were determined by BioSpec-nan (Shimadzu, Japan). Total RNA (1 μg) was reversely transcribed into cDNA using FastKing Reverse Transcriptase Kit (Tiangen, China). cDNA samples were used to amplify the target genes using SuperReal PreMix Plus (SYBR Green) kits (Tiangen, China) on an Eppendorf AG 22331 Real-Time PCR system (Eppendorf, Germany). Specific primers (Table 1) were synthesized by Comate, China. GAPDH expression in each sample was included as the internal control. Gene expression was quantitated using the 2^−∆∆Ct method.

Materials from different developmental stages of P. sojae, including mycelia (MY), sporulating hyphae (SP), zoospores (ZO), cystospores (CY), germinated cystospores (GC), and the process of infection (at 1.5, 3, 6, 12, 24, and 48 h), were collected as described previously [29 (link)]. Total RNA was extracted using the SV Total RNA Isolation kit (Promega, Madison, WI, USA) following the recommended protocol, and RNA quality was tested via agarose gel electrophoresis. First-strand cDNA was synthesized from 1 μg of total RNA using the FastKing Reverse Transcriptase kit (Tiangen, Beijing, China). Quantitative real-time PCR (qRT-PCR) was performed on a Bio-Rad CFX Connect Real-Time PCR System using SuperReal qPCR Mix (Tiangen) following the manufacturer’s instructions. Actin sequence from P. sojae was used as a reference gene, and the primers for this experiment are listed in Table S1. Relative PsZFPK1 transcript levels were calculated using the 2^−ΔΔCT method [30 (link)]. Means and standard deviations were calculated using data from three biological replicates.