RNA Extraction and qRT-PCR Analysis from Fruit Samples

RNA extraction from 70 mg fruit samples was made using the Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada), modified with the addition of 2% PVP and 2% β-mercaptoethanol to the lysis buffer. Purified RNA was quantified by Qubit (Invitrogen, Carlsbad, CA, USA) fluorometry and integrity was checked by agarose electrophoresis. RNA was reverse transcribed to cDNA in a total volume of 10 μL, using the PrimeScript RT Reagent Kit (Takara Bio, Otsu, Japan).
The qRT-PCR was performed on a StepOnePlus Real-Time PCR System (LIFE TECHNOLOGIES, Carlsbad, CA, USA), using 1 µL of 10X diluted cDNA, SYBR premix Ex Taq (Tli RNaseH plus) (TAKARA BIO, Kusatsu, Japan) [50 (link)]. DkTUA was used as a housekeeping gene reference [51 (link)]. Gene-specific primers used for expression analysis are listed in Table S2. Primers were designed using coding sequences from NCBI genes and Primer3 software v. 4.1.0 (https://primer3.org) accessed on 10 November 2020. The specificity of the designed primers was checked by the presence of a single peak in the dissociation curve after the amplification.