Quantitative RT-PCR Analysis of Grapevine Transcripts

Total RNA from the grapevine was extracted using the method described by Reid et al. [40 (link)]. The integrity of extracted RNA was checked on 1% agarose gels to ensure that the 28S and 18S were clear without tailing and that the 28S:18S ratio was 2:1. The RNA concentration was determined using an Ultramicro Spectrophotometer (P-330-31, Implen, Munich, Germany). The cDNA first strand was synthesized from 1 µg total RNA using the PrimerScript^TM II 1st Strand cDNA Synthesis Kit (Takara). Quantitative RT-PCR was run on IQ5 real time PCR cycler (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR^® Green I dye. Reactions were the following thermal profile: 3 min at 94 °C, 40 cycles of 5 s at 94 °C, 30 s at 58 °C. The relative mRNA rations were calculated as 2^−ΔΔCT [41 (link)]. The transcript levels of target genes were normalized against VvActin for the grape samples and NtActin for Nicotiana samples [40 (link),42 (link)]. The data are presented as the mean value of three biological replicates.