RNA Extraction and RT-PCR Analysis

RNA was isolated from callus tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA), as described in a previous study [20 (link)]. Reverse transcription reactions were performed by adopting the SuperScript First-Strand Synthesis System (Invitrogen), as described in a previous study [26 (link)]. Messenger RNA expression was calculated as a ratio relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and expressed relative to V-WT group levels. In brief, real time polymerase chain reaction (RT-PCR) analyses were performed using the LightCycler System (Roche, Indianapolis, IN, USA) to determine the number of cDNA molecules in the reverse transcribed samples. PCR was performed using 2-mL LightCycler DNA Master SYBR Green I (Roche), 0.25 mM of 5′ and 3′ primers, and 2-mL samples or H₂O to a final volume of 20 mL. The concentration of MgCl₂ was 3 mM. Sample denaturation, amplification, and fluorescence determination were carried out as described in a previous study [20 (link)]. Purified PCR fragments of known concentration were serially diluted and served as external standards for each experiment to determine the number of copies of the targeted DNA in the samples. GAPDH levels in the samples were used to normalize the data. The primer sequence used for the real-time PCR was the same as that described in previous studies (Table 1) [20 (link),31 (link)].