Abi quantstudio 3 real time system

The Trizol reagent (Invitrogen, USA) was adopted for total RNA isolation of all collected samples referring to manufacturer’s protocol. 2 μg of extracted RNA was measured for reverse transcription into cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). Potato 14-3-3s specific primers for this experiment were designed through Primer Express 3.0 software (Applied Biosystems), and the specificity of all primers were further verified in NCBI database with Primer Blast program (Table S3). The EF1α (Elongation factor 1α) gene was served as an internal control (Charfeddine et al., 2015 (link)). The qPCR was performed on an ABI Quant studio 3 Real-Time system (Applied Biosystems) using SYBR^® Green Realtime PCR Master Mix (TOYOBO, Japan) with the reaction program: The program as follows: denaturation (95°C for 5min), amplification and quantification (40 cycles of 95°C for 15 s and 60°C for 1 min), melting curve analysis (60–95°C, with a heating rate of 0.3°C/s). The comparative delta cycle threshold (DDCT) method was adopted to calculate relative transcript levels of 14-3-3s (Wang et al., 2017 (link)). Statistical analysis was performed by one-way analysis of variance (ANOVA) test using SPSS 19.0 software (http://www.spss.com.cn/ ). Each qPCR assay was established with three technical replicates.

To verify the reliability of transcriptome sequencing, quantitative real-time transcription PCR (qRT-PCR) was performed. Fourteen DEGs were selected, and the primers (Additional file 4) of these genes for qRT-PCR were designed using primer premier 5.0 [51 (link)]. The qRT-PCR reaction was executed using the TB Green Premix Ex Taq II (TaKaRa, Beijing, China) and carried on ABI QuantStudio®3 Real-Time System (Applied Biosystems, CA, USA). The amplification procedure was started with an initial denaturation at 95℃ for 10 min, followed by 40 cycles of 95℃ for 15 s, and 60℃ for 1 min. The α elongation factor [52 (link)] was used as an internal control for qRT-PCR amplification, and all reactions were set in triplicate. The relative 2^−△△Ct method was used to determine the expression levels of the 14 tested genes [53 (link)]. Pearson correlation analysis was performed between the data of qRT-PCR and transcriptome sequencing by cor.test in R.

Total RNA was isolated from all collected samples using Trizol reagent (Invitrogen, USA) according to the manufacturer ‘s protocol. Extracted RNA (1 μg) was reverse transcribed into cDNA using the PrimeScript™ RT kit with gDNA Eraser (TaKaRa, Japan). Primer Premier 5.0 was used to design the specific primers and Primer Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) program was used to further verify the specificity of all primers in the NCBI database. qPCR was performed on an ABI Quant studio 3 RealTime system (Applied Biosystems, USA) using SYBR ^® Green RealTime PCR Master Mix (Novoprotein, China) with the following reaction program: denaturation (95°C 30 s), amplification and quantification (95°C 3 s, 60°C 30 s, 40 cycles), and melting curve analysis (60–95°C, heating rate 0.3°C/s). Actin gene was used as internal reference. The relative expression levels of genes were calculated by the 2^−ΔΔCT method. Statistical analyses were performed using Student’s t-test (t-test), with three technical replicates established for each qPCR method. Relevant figures were drawn using TBtools and prism 8.0.