Primescript rt pcr reagent kit

A total of 50–100 mg of frozen berry samples representing the different development stages was ground in a mortar and the resultant powder was used for the RNA extraction with the TRIzol extraction reagent (Invitrogen, Carlsbad, CA, USA). Agarose gel electrophoresis was used to assess the quality of every RNA sample and high-quality RNA samples with an A260/A280 ratio of 1.8:2.1 were used as the template material for the qRT-PCR analyses. Specifically, complementary DNA (cDNA) was transcribed using the Takara Prime Script TM-RT PCR reagent Kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. Since the monoterpene volatiles showed the most significant variations after the elicitor treatment, we quantified the expression of genes in the terpenoid metabolic pathways by qRT-PCR analysis (ABI7300, Applied Biosystems, Milan, Italy). Relative transcript levels were calculated using the 2^−ΔΔCt method [53 (link)] and VvActin was used as the reference for normalization purposes. All reactions, including non-template controls, were performed in triplicate. Sequences of the primers used to amplify the VvDXS1, VvDXS3, VvDXR and VvHDR genes were referenced from Martin [43 (link)]; VvPNGer and VvPNlinNer1 were referenced from Matarese [47 (link)].

Expression pattern verification of 8 randomly selected DEGs from RNA-Seq was performed using qRT-PCR, as described previously (Zhu et al., 2019 (link)). Complementary DNA (cDNA) was transcribed using the Takara Prime Script TM-RT PCR reagent Kit (Takara, Japan) according to the manufacturer’s instructions. Specific primers (Supplementary Table 1) for qRT-PCR were designed using the Primer Premier 5 software. Actin gene was selected as internal reference in this study. qRT-PCR was conducted using the ABI7500 RT-PCR system according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, United States). All reactions and non-template controls were performed in triplicate. Relative transcription levels were calculated using the 2^–ΔΔCt method (Livak and Schmittgen, 2001 (link)). Data are expressed as mean ± standard error (SE) (n = 3).

Total RNAs were extracted from 2-week-old plants of the wild-type and atmyb44 mutant plants using the RNAiso Plus reagent from Takara (Otsu, Japan). The cDNA was synthesized from 1 μg of total RNAs with PrimeScript RT-PCR reagent Kit of Takara. qRT-PCR was performed using the CFX96 real-time PCR detection system from Bio-Rad Laboratories (Hercules, CA, USA) and SYBR Premix Ex Taq II from Takara (Otsu, Japan). The relative expression levels were calculated using the ΔΔC_t method [43 (link)], which was normalized against the expression level of ACTIN2/8. For all qRT-PCR analyses, three technical replicates and three independent biological repetitions were performed. Statistical significance was assessed by Student’s t-test. Values of p < 0.05 were considered significant. Primers used for this assay are listed Table S2.