Trizol up plus rna kit

Based on the CsKCS gene sequences screened from transcriptome data, qRT-PCR (quantitative reverse transcription PCR) primers (Table S5) were designed online using IDT (https://sg.idtdna.com/Pages, accessed on 25 January 2023). The primer sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. The total RNA was extracted using a TRIzol Up Plus RNA Kit (TianGen Biochemical Technology Co., Ltd., Beijing, China), and its quality and concentration were detected by a 1.0% agarose (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) gel electrophoresis and nucleic acid concentration detector (Implen (Beijing) International Trading Co., Ltd., Beijing, China). The RNA was reverse transcribed into cDNA by using the Tiangen Fastking gDNA Dispelling RT SuperMix kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). A real-time qRT-PCR assay was performed on a Bio-Rad CFX Connect^TM real-time quantitative PCR instrument (Bio-Rad, Hercules, CA, USA). The qRT-PCR reaction system consisted of 10 μL SYBR Green qPCR Mix, 0.4 μL upstream and downstream primers, 1.5 μL cDNA template, and ddH₂O to 20 μL. Reaction procedure: 95 °C for 3 min; 40 cycles of 95 °C for 10 s and 60 °C for 20 s; 72 °C for 30 s, with CsGAPDH as the reference gene. The 2^−ΔCt algorithm was used to calculate the gene expression [55 (link)], and the heat map was drawn by TBtools software.

The total RNA was extracted using a TRIzol Up Plus RNA Kit (TianGen Biochemical Technology Co., Ltd., Beijing, China), following the manufacturer’s instructions. The RNA quality was checked by electrophoresis on a 1.0% (w/v) agarose gel. The cDNA was synthesized by reverse transcription using a Prime Script^TM II 1st strand cDNA Synthesis Kit (Beijing Solarbio Technology Co., Ltd., Beijing, China).

For reverse transcription and quantitative real‐time PCR, total RNA was isolated from different tissues using the Trizol Up Plus RNA Kit (Tiangen Biotech). cDNAs were synthesized with Superscript II reverse transcriptase (TransGen Biotech). The qRT‐PCR was performed using the ABI7900 system (Applied Biosystems). The PCR reactions contained 1 μL of 1:5 diluted cDNA, 0.2 μL of gene‐specific primers, 0.2 μL of 50× ROX High Reference Dye, 5 μL of 2 ×KAPA SYBR^® FAST qPCR Master Mix (KAPA Biosystems) and water to a final volume of 10 μL. The PCR cycling conditions were as follows: 95 °C for 30 min followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Three biological replicates were performed for each reaction, and the qRT‐PCR data were analysed using SDS2.3 software. The primers used for real‐time quantitative PCR are listed in Table S8.