Reverse transcription kit

The RNA was extracted from samples of six developmental stages of plum using the Tiangen kit and was reverse transcribed to cDNA using the Vazyme Reverse Transcription Kit (Vazyme, Nanjing, China, code: R212). The RT-qPCR analysis was performed on an ABI QuantStudio 1 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR green (Vazyme Biotechnology, Nanjing, China) in a 10 μL reaction mixture containing 1 μL diluted cDNA, 10 μM of each primer 0.4 μL, ddH₂O 3.2 μL, and 5 μL 2 × AceQ Universal SYBR qPCR Master Mix. The PCR reaction protocol was 95 °C for 2 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. The qRT-PCR results were calculated using the 2^−ΔΔCT method. Three biological replicates were established for each sample. The CAC and Ubi genes were used as housekeeping genes in Prunus salicina and pepper, respectively [2 (link)]. All the primers used in the PCR analysis are listed in (Table S1).

The RNA was extracted from samples of six developmental stages of plum using the Tiangen kit and was reverse transcribed to cDNA using the Vazyme Reverse Transcription Kit (Vazyme, Nanjing, China, code: R212). The RT-qPCR analysis was performed on an ABI QuantStudio 1 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR green (Vazyme Biotechnology, Nanjing, China) in a 10 μL reaction mixture containing 1 μL diluted cDNA, 10 μM of each primer 0.4 μL, ddH₂O 3.2 μL, and 5 μL 2 × AceQ Universal SYBR qPCR Master Mix. The PCR reaction protocol was 95 °C for 2 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. The qRT-PCR results were calculated using the 2^−ΔΔCT method. Three biological replicates were established for each sample. The CAC and Ubi genes were used as housekeeping genes in Prunus salicina and pepper, respectively [2 (link)]. All the primers used in the PCR analysis are listed in (Table S1).

Sixty-three calf diarrhea samples from Shijiazhuang, Heibei Province, manifesting as weakness, fever, excretion of watery feces, and accompanied by a foul smell, were subjected to RNA extraction using HiPure Total RNA Mini Kit (AnGen, Guangzhou, China). The total RNA was further reverse transcribed into cDNA using a Vazyme Reverse Transcription Kit (Vazyme, Nanjing, China). PCR amplification was performed using 2× Taq Master Mix (Vazyme, Nanjing, China) with the following primers: For BVDV: sense, 5′-AGCGGGGATAAGGTTGGAAA-3′; antisense, 5′-ACCTGCAGCCCCTTTTCTAT-3′. For BCoV: sense, 5′-GCTACCAATTATTTTGCTTGGC-3′; antisense, 5′-ATGGAGAGGGCACA GACTTATC-3′. For BoAstV: sense, 5′-GAYTGGACBCGHTWTGATGG-3′; antisense, 5′-K YTTRACCCACATNCCAA-3′. For BRoV: sense, 5′-ACCACCAAATATGACACCAGC-3′; antisense, 5′-CATGCTTCTAATGGAAGCCAC-3′ (Genscript, Nanjing, China). The above four pairs of primers were designed with reference to sequences GenBank accession no. KF501393, LC494178, LC047787, and MZ540977, respectively, using the Primer Premier 5.0 software package. The PCR reactions were performed with 2 µL of cDNA templates added to 18 µL of reaction mixture, containing final concentrations of 0.5 µM of each primer. The cDNA was amplified by 35 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 35 s, and extension at 72 °C at 1 kb/min, and finally extended at 72 °C for 10 min.

SPL genes were identified according to the functional annotation results. The expression of the SPL gene in the transcriptome was analyzed based on the FPKM values.
To confirm the expression of these SPL genes, a real-time quantitative PCR (qRT-PCR) was performed using gene-specific primers (Table S6, in Supplementary Materials S1). Actin was selected as the reference gene. For either the juvenile or adult leaf samples, three biological replicates were mixed to extract the RNA. The extraction method was the same as that reported in Section 2.2.2. Then, cDNA was obtained according to the instructions of the Vazyme Reverse Transcription Kit (Vazyme, Nanjing, China). The reaction was performed with a Bio-Rad CFX96™ real-time PCR instrument (Bio-Rad, Hercules, CA, USA) using the following procedure: pre-denaturation at 95 °C for 30 s; 40 cycles of 95 °C for 5 s and 60 °C for 30 s; 95 °C for 10 s, 65 °C for 5 s, and 95 °C for 5 s. All qRT-PCR experiments were performed with three technical repetitions. The relative expression levels of the SPL genes were calculated using the 2^−ΔΔCT method [69 (link)]. All data were expressed as mean ± standard error (SE), and the Student’s t-test was used for statistical analysis.

Total RNA was isolated from the cells using TRIzol reagent (Thermo Fisher, Cat. #15596026). Reverse transcription kit (Vazyme, Cat. #R211-01) and ChamQ Universal SYBR qPCR Master Mix (Cat. #Q711) were used for quantitative PCR of the target genes according to the manufacturer's instructions. RT-qPCR primer sequences are listed in Table S2. GAPDH gene expression was used as the endogenous control. The relative expression of the target genes relative to the control was calculated according to the 2-ΔΔCT formula. Each experiment was performed in triplicate.

Total RNA was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit (RC101-01, Vazyme, China) with on-column DNA digestion. The reverse transcription was performed using a reverse transcription kit (R333-01, Vazyme, China), with the reaction system containing 4 µL of 5 × qRT SuperMix II, 1 µg of total RNA, and RNase-free ddH₂O to a final volume of 20 µL. The reaction program was set as follows: 15 min at 50 °C, 5 s at 85 °C, and then storage at 4 °C. The primers for qRT-PCR were designed using Primer Premier 5.0 and are summarized in Table S5. GAPDH was used as control. All qPCR reactions were conducted in a 10 µL reaction volume with 1 µL of cDNA, 0.2 µL of each primer (10 µmol/L), 5 µL of 2 × AceQ Universal SYBR qPCR Master Mix, and 3.6 µL of ddH₂O. Thermocycler settings were as follows: 95 °C for 5 min, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Three replicates were conducted for all analyses.