Quantification of UFGT Gene Expression in Berry Skin

Total RNA was isolated from frozen berry skin samples per the protocol described by Boss et al. (1996) (link). DNA was removed from samples using RNase-free RQI treatment per the manufacturer’s instructions (Promega, Madison, WI, USA) followed by a cleanup with the RNeasy mini kit (Qiagen, Valencia, CA, USA). cDNA was synthesized from total RNA using the qScript™ cDNA Synthesis kit (Quanta Biosciences, MD, USA). Quantitative real-time expression was performed using PerfeCTa SYBR Green SuperMix ROX (Quanta Biosciences, MD, USA) on the ABI7300 real-time system (Applied Biosystems, CA, USA) and using UDP-glucose: flavonoid-3-O-glucosyltransferase (UFGT) gene’s primer designed with the Primer Express 2.0 software (Applied Biosystems) as described in (Afifi et al., 2021 (link)). Relative quantification for UFGT gene was calculated by the 2–ΔΔT method (Livak and Schmittgen, 2001 (link)) using the grape ubiquitin gene as a constitutive control (Fujita et al., 2005 ). The ubiquitin primer was designed from a tentative consensus sequence TC38636 and described in (Afifi et al., 2021 (link)). QRT-PCR analyses were performed using three biological replicates using sets of cDNA from independent samples. Gene expression was calculated as an increase/decrease fold change in reference to the sample collected from the untreated treatment.