Perfectstart green qpcr supermix kit

After processing the beef tissues, we used RNAiso Plus Kit (Trizol, Takara, Beijing, China) to extract the total RNA from the beef heart, liver, spleen, lung, kidney, muscle, and adipose tissues. The cDNA was obtained by reverse transcription kit (PrimeScript™ RT reagent Kit with gDNA Ewraser, Takara, Beijing, China). The DNA and CDS region sequences of hub genes were downloaded from the NCBI database for primer design. Then, the designed primer sequences were uploaded to BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) for specificity test. Primer sequences are shown in Table 1. The relative expression levels of hub genes in adult cattle heart, liver, spleen, lung, kidney, muscle, and adipose tissue were measured, and the expression levels of hub genes in the adult cattle and the newborn calves' adipose tissue were compared. Quantitative real-time PCR were performed using the PerfectStart Green qPCR SuperMix kit (TransGen Biotech, Beijing, China), and the results were obtained. It should be noted that three biological replicates and technical replicates were performed for all experiments. SPSS 25 (37 (link)) and Graphpad Prism 9 (38 (link)) softwares were used for difference significance analysis and mapping, respectively.

Total RNA was extracted using an RNAprep Pure Plant Kit (Tiangen), following the manufacturer’s protocols. GAPDH was used as an internal reference gene [73 ], and Primer Premier 5.0 was used to design primers for the target gene (Supplementary Data Table S3). qRT–PCR was performed on a CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA) using a PerfectStart Green qPCR SuperMix Kit (TransGen Biotech Co. Ltd.). Relative gene expression was calculated using 2^−ΔΔCt [74 (link)].

For fluorescence quantification of tobacco leaves, the relative intensities of SMV-GFP/TMV-GFP were processed and quantified with software Image J v1.8.0 (National Institutes of Health, Bethesda, MD, USA). The fluorescence intensity of each gene was subjected to 10 technical replicates. For virus titer detection, total RNA was isolated from mixed samples of corresponding infection areas of 10 individual leaves and first-strand cDNA was synthesized after co-infiltrating tobacco leaves with different viral infectious clones and candidate genes for 4 days. The control group (EV) used pCambia1300 empty vector and corresponding viral infectious clones for mixed infection, and their antiviral activity was determined by comparing the accumulation level of the virus after adding the genes we screened. RT-qPCR was performed using the Applied Biosystems ViiA 7 using PerfectStart Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China, TG-AQ601) following the manufacturer’s instructions. Three biological replicates were performed for each experiment. Graphs were generated by GraphPad Prism 8 (La Jolla, CA, USA), Excel 2019 (Microsoft, Redmond, WA, USA). Error bars represent the SD of the mean, and significance was indicated when p < 0.05.

Total RNA of Day 10 nematodes treated as above was extracted using TransZol Up Kit (TransGen Biotech) and reverse‐transcribed with EasyScript® All‐in‐One First‐Strand cDNA Synthesis Kit (TransGen Biotech) according to the manufacturer's instructions. Quantitative real‐time PCR was carried out on a StepOnePlus Real‐Time PCR Instrument (Applied Biosystems) using PerfectStart^® Green qPCR SuperMix Kit (TransGen Biotech) with act‐1 as a reference gene. All reactions were performed in three technical replicates from three biological repeats. The primers of gene transcripts used for the PCR analysis are listed in Table S9.

The total RNA was isolated from the seedlings or young leaves using Plant RNA kit (Omega, United States) according to the manufacturer’s instructions. Approximately 1 μg of total RNA was reversely transcribed into cDNA using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen, Beijing, China) according to the manufacturer’s instructions. qRT-PCR was used to measure transcript levels of CgbHLH001 and stress-related genes. The primers were shown in Supplementary Table 1. qRT-PCR analysis was performed in a LightCycler 96 Real-Time System (Roche, United States); the PCR reaction conditions were as follows: 94°C 30 s; 40 cycles of 94°C 5 s, 60°C 30 s. qPCR was performed with PerfectStart Green qPCR SuperMix kit (TransGen, Beijing, China). Three biological replicates with two technical replicates of each were applied to each treatment, and the 2^–ΔΔCT method (Shi and Chiang, 2005 (link)) was employed to calculate the relative expression level of each gene. Ntactin was used as an internal reference for tobacco to normalize the expression level. The relative quantification was described as fold change of gene expression in the test sample compared to the control.