Transstart green qpcr supermix udg

To assess the plant defense mechanisms induced by the exogenous foliar application of PeBb1 elicitor on B. rapa plants, relative expression of key genes associated with B. rapa’s ET and JA pathways (Table 2) were examined by real-time quantitative PCR (RT-qPCR). Using the EasyPure^® Plant RNA Kit (TransGen Biotech, Beijing, China), total RNA was extracted from the leaves of aphid infested protein-treated and buffer-treated (control) plants of B. rapa. TransScript^® All-in-One SuperMix for qPCR Kit (TransGen Biotech, Beijing, China) was used to synthesize first-strand cDNA. The relative expression levels of both type of defense-related genes were determined by RT-qPCR performed on ABI 7500 Real-Time PCR System thermocycler (Applied Biosystems, Foster City, CA, USA) using TransStart^® Green qPCR SuperMix UDG (TransGen Biotech, Beijing, China). Three independent biological and three technical replicates were performed for each sample. Actin was used as a quantitative control. Relative expression levels were calculated using the 2^–ΔΔCT method [44 (link)]. Primer pairs used for the amplification of these plant defense associated genes are detailed in Table 3.

RT-qPCR was performed according to the method described by Liu et al. [21 (link)], with β-Actin as the reference gene, the genes on the MPs and citrinin gene cluster were selected, and the expression of these genes was detected by qRT-PCR during the submerged fermentation of M. purpureus LQ-6 and M. purpureus CSU-M183, respectively. For removal of residual genomic DNA, RNA samples were treated with RNase-free DNaseI (Thermo Fisher Scientific, Massachusetts, USA) following the manufacturer’s protocol. The first-strand cDNA was synthesized using oligo-dT primers and EasyScript Reverse Transcriptase (TransGen Biotech, Beijing, China), according to the manufacturer’s protocol. qRT-PCR was performed using the TransStartGreen qPCR SuperMix UDG (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. 2^−ΔΔCT was used to determine expression levels of the tested genes. The primers used in these analyses were listed in S1 Table.

Quantitative reverse transcription-PCR (qRT-PCR) was performed to determine the relative expression levels of selected transcripts. The total RNA samples were utilized for gDNA-free cDNA synthesis with the ReverTra Ace-ɑ-® kit (Cat # FSK-101, TOYOBO, Japan). The synthesized cDNA was used as the template for PCR amplification of the selected genes with their corresponding primer pairs (Supplementary Table 1). The B. cinerea tubulin gene (BC1G_05600) was used to correct for sample-to-sample variation in the amount of RNA (Dulermo et al., 2010 (link)). Amplification was carried out by the CFX96 Real-Time PCR Detection System (BioRad, United States), using the TransStart Green qPCR SuperMix UDG (Transgen, China). The relative abundances of selected transcripts were calculated by the 2^−ΔΔCt method from the mean of three independent determinations of the threshold cycle (Schmittgen and Livak, 2008 (link)).

To analyze whether FocCP1 exhibited elicitor function, 4-week-old tobacco leaves were infiltrated with FocCP1 (100 μM) with BSA (100 μM) as a control. The samples were cryo-preserved in liquid nitrogen post-treatment of 8, 16, and 24 h, three biological replicates were performed [15 (link)]. All total RNA was extracted with the RNA Prep-Pure Plant Kit (TianGen, Beijing, China) according to the manufacturer’s protocol. First-strand cDNAs were synthesized with the same method as in Section 4.4. Six defense-related genes, PR1, PR5, PAL, EDS1, HSR203J, and LOX, were amplified using an ABI 7500 Real-Time PCR system (Applied Biosystem, Foster CA, USA) with Trans Start^® Green qPCR Super Mix UDG (TransGen Biotech, Beijing, China). Tobacco actin gene was the endogenous control and the qRT-PCR primers are listed in Table 3 [67 (link)]. The amplification program was as follows: 50 °C for 2 min, 94 °C for 10 min, followed by 40 cycles of 94 °C for 5 s, 60 °C for 15 s, and 72 °C for 15 s. The relative mRNA expression values were calculated according to 2^−ΔΔCt methods [68 (link)]. For each gene, qRT-PCR assays were repeated three times with three biological replicates each time.

RNAs were extracted from the samples using (Promega, China) according to the manufacturer’s specifications. The first-strand cDNAs were synthesized by reverse transcription of 100 μg total RNA which was generated using the Easy Script First-strand cDNA Synthesis SuperMix Kit (TransGen Biotech, China). Synthesized cDNA was diluted 1:10 with nuclease-free water for use in qRT-PCR. The expression levels of the genes were normalized to the sorghum housekeeping gene GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDH) gene as an internal control. Gene-specific primers were designed using the National Center for Biotechnology Information (NCBI) Primer-BLAST tool and were synthesized by Sangon (Beijing, China). Primers used in this study are listed in Supplementary S7 File. qRT-PCR was carried out using TransStart Green qPCR SuperMix UDG (TransGen Biotech, China) following the manufacturer’s instructions, on a Bio-Rad CFX96TM real-time PCR detection system (Bio-Rad, USA). Reaction parameters for thermal cycling were as follows: 94°C for 10 min, 40 cycles of 94°C for 5 sec and 55°C for 15 sec, 72°C for 10 sec. We performed RT-qPCR on three biological replicates and used the 2^–ΔΔCt method for quantification [39 (link)].

The cDNA was synthesized from 5 μg of RNA using the ThermoScrip RT-PCR System (Invitrogen). Real-time PCR was performed in duplicate for each RNA sample using the TransStart™ Green qPCR SuperMix UDG (TransGen Biotech) with an appropriate cDNA dilution as a template. Three biological replicates were performed for each strain. Control reactions were carried out in parallel in the absence of the reverse transcriptase, 16S rRNA was used as an internal standard to normalize the expression levels of the tested sRNA candidates. Relative quantitative analysis was performed across different cDNA templates using the LightCycler 480 software (Bio-Rad).