Plant fungi total rna purification kit

Stem and/or leaf tissues from each plant sample were placed in individual plastic bags (Bioreba, Reinach, Switzerland) with an extraction buffer (PBS containing 0.2% of diethyldithiocarbamate and 2% of polyvinylpyrrolidone-10) in a ratio of 1:5 (w:v). The samples were grinded with Homex 6 (Bioreba, Reinach, Switzerland).
Total RNA was purified from 200 μL of plant extract using a commercial Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corporation, Thorold, ON, Canada) following the manufacturer’s instructions. The RNA was eluted in a total volume of 50 μL and quantified using a DeNovix DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA). All RNA purifications were stored at −80 °C until subsequent analysis.
The RNA from mealybugs was purified using a Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corporation, Thorold, ON, Canada) with slight modifications. Briefly, the mealybugs were placed in tubes containing 250 μL of a lysis buffer and a similar volume of 212–300 μm glass beads (Sigma-Aldrich, Burlington, MA, USA). The tissue was homogenized, shaken in a vortex for 2 min and centrifuged for 5 min at 10,000× g. The supernatant was transferred to a fresh tube and the extraction procedure was followed as indicated by the manufacturer.

Total RNA was extracted using the Plant/Fungi Total RNA Purification kit from Norgen Biotek Corporation (Thorold, Canada). It was quantified using the Qubit^(R) RNA BR Assay kit provided by Life Technologies (Carlsbad, United States). The quality of the RNA was verified on a 1% agarose gel.

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.