Sybr premix ex tag

Total RNA was extracted from the tea seedling with TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRA), and the corresponding cDNA was obtained with cDNA Reverse Transcription Kit (TaKaRa). Then qPCR was conducted with SYBR Premix Ex Tag on a DNA Engine Opticon^TM 2 system (PCR instrument: Bio-rad T100, manufacturer: Bio-Rad Laboratories Inc, city: State of California, Country: United States). The CsTBP (TATA-box binding protein gene)gene was used as a housekeeping gene to normalize the expression level of target genes in C. sinensis. All primers used in this study are listed in Supplementary Table S10. The reaction conditions of the PCR program were as follows: an initial step at 95 °C for 30 s. After the cycling protocol, melting curves were obtained by increasing the temperature from 60 to 95 °C (0.2 °C^−s) to denature the double-stranded DNA. Then qPCR amplifications were carried out in 96-well plates. The assays were run in an ABI 7500 system, using the SDS v. 1.4 application software (Applied Biosystems, Foster City, CA, USA). The primer efficiency was created based on a five-fold dilution series of cDNA (1:5, 1:25, 1:50, and 1:100), followed by 42 cycles of 95 °C for 15 s, 58 °C for 15 s, 72 °C for 30 s, and 1 s at 80 °C for reading the plate. The expression levels were evaluated by the 2^−ΔΔCt method, with three biological replicates [71 (link)].

Total RNA was extracted from leaves of T1 transgenic plants using the RNAasy kit (Qiagen, Inc., Hilden, Germany) as per the manufacturer’s protocol. First-strand cDNA was synthesized using the PrimerScript RT Reagent Kit (TaKaRa, Beijing, China). qPCR was performed with the SYBR Premix ExTag on the Bio-Rad CFX96 (Bio-Rad, Shanghai, China), with the SlActin gene as internal control. The qPCR primers were designed using Primer Premier v6.0 (Palo Alto, CA, USA) (YG Soly720-F: 5′-CTTTCGATTCACTCGTGGGAT-3′; YG Soly720-R: 5′-GCCGAACCTAAACCTGTGCT-5′). Gene expression levels were calculated using the 2^−ΔΔCT method [39 (link)].

Total RNA of transgenic poplar plants was extracted using a Quick RNA Isolation Kit (Huayueyang, Beijing, China). The total RNA was treated with DNaseI according to the manufacturer’s instructions. One microgram of total RNA of each sample was used as a template for cDNA synthesis using a SuperScript gDNA Removal cDNA Synthesis Kit (Huayueyang). For quantitative real-time reverse transcriptase–PCR (RT-qPCR), SYBR Premix Ex Tag was used on a CFX Connect Real-Time PCR system (Bio-Rad, Shanghai, China).
The qRT-PCR reactions were conducted in 96-well plates. The melting temperature of the products was determined to verify the specificity of the amplified fragments. The primers used for qRT-PCR are listed in Supplementary Table S2. The results were analyzed by the ΔΔ^CT method using PtActin as the reference gene. All the qRT-PCR data points were established as three biological replicates, and three technical replicates were conducted.