Cfx 96 well real time pcr system

For endpoint PCR analysis intron-spanning primers specific for human frizzled receptor 1–9 and β-actin were used (Table 1). Total RNA was isolated using Trizol (Invitrogen), and 2 µg of RNA was reverse-transcribed with M-MLV Reverse Transcriptase (Promega). Endpoint PCR reactions utilized JumpStart RedTaq (Sigma) on Veriti thermocycler (Applied biosystems). Real-time PCR Reactions for were ran on CFX96 well Real-Time PCR System (Bio-Rad) and utilized PerfeCTa SYBR green FastMix, ROX (Quanta Bioscience, Inc., MD, cat. number 95073-012).

Total RNA was extracted as above, and the purity and concentration were measured by a NanoDrop 2000 spectrophotometer (Thermo Scientific Co., Ltd., Wilmington, NC, USA). The first-strand cDNA was synthesized using the Prime-Script^® RT reagent kit (Takara, Dalian, Liaoning, China) according to the manufacturer’s guidelines. qPCR was performed using SYBR^® Premix Ex Taq II (Takara Co., Ltd., Dalian, Liaoning, China) on a CFX-96-well real-time PCR system (BioRad Co., Ltd., Hercules, CA, USA). The relative expression levels of target genes were calculated using the 2^-−ΔΔCT method and normalized using EF1α and actin reference genes in P. bournei and N. tabacum, respectively. The primers of target genes and reference genes are listed in Supplementary Table S2. Three replicates were performed for each sample.

Total RNA was extracted using the M5 Plant RNeasy Complex Mini Kit (MF045, Mei5bio, Beijing, China) and reverse transcription was performed using the HiScript III All-in-one RT SuperMix Perfect for qPCR (R333, Vazyme, Nanjing, China). Quantitative real-time PCR (qRT-PCR) was carried out using the ChamQ SYBR Color qPCR Master Mix (Q411, Vazyme, Nanjing, China) on a CFX-96-well real-time PCR system (Bio-Rad, Hercules, CA, USA). The qRT-PCR reaction procedure was a two-step amplification method: pre-denaturation phase 95 °C for 30 s; cyclic reaction phase 95 °C for 10 s and 60 °C for 30 s, repeated for 40 cycles; melting phase 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The fold change in target genes was calculated as described in [40 (link)], with PbEF1α as an internal reference gene. The primers are listed in Table S9.