Cfx connect real time pcr system

In brief, the cells were lysed by TRIzol® reagent (Invitrogen, 15596018), followed by sequential RNA precipitation by isopropanol and ethanol. After air drying, the RNA pellet was redissolved in an appropriate volume of DNase/RNase-free H₂O. To obtain complementary DNA (cDNA), 1 μg of total RNA was reverse transcribed by using iScript™ Reverse Transcription Supermix (Bio-Rad, 1708841) according to the manufacturer’s protocol. The relative expression of each gene was measured in a Bio-Rad CFX Connect™ Real-Time PCR System by using the SYBR Green method (Tiangen, FP205-02). The expression of human or mouse β-Actin was used as an internal control.

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.