Ssoadvanced universal inhibitor tolerant sybr green supermix

A total of 200 mg of tissue were macerated in liquid nitrogen with a mortar and pestle for DNA extraction using the Plant DNA Purification Kit (Norgen BIOTEK Corp., Thorold, ON, Canada), according to the manufacturer’s instructions. Detection of CLas was achieved by RT-qPCR assay using primers HLB-4G (5′AGTCGAGCGCGTATGCGAAT-3′) and HLBr (5′-GCGTTATCCCGTAGAAAAAGGTAG-3′) following the protocol reported by [72 (link)]. Amplifications were performed using a thermocycler CFX-96 Real-Time PCR System in a 96-well PCR plate (Bio-Rad, Hercules, CA, USA) and SsoAdvanced Universal Inhibitor-Tolerant SYBR^® Green Supermix (Bio-Rad) for signal detection. All reactions were performed in triplicate in 10 µL reaction volumes, using 200 ng of DNA per reaction. Plants were considered PCR-positive for CLas when the CT (cycle threshold) value was below 34.

Total RNA was extracted from the respective latent cell lines using the RNeasy Plus Mini Kit (QIAGEN; Cat # 74134), and its concentration and purity were assessed using Nanodrop. 2 μg of total RNA from each sample were subjected to cDNA synthesis using the iScript^™ Advanced cDNA Synthesis Kit (BIO-RAD; Cat # 1725037) following the manufacturer’s instructions. The qRT-PCR reaction mixture contained 5 μL SsoAdvanced^™ Universal Inhibitor-Tolerant SYBR^® Green Supermix (BIO-RAD; Cat # 1725016), 0.5 μL gene-specific forward or reversed primer (See the Table of primers below), 4 μL nuclease-free water (Sigma; Cat # W4502-50ML), and 0.5 μL cDNA (1:10 diluted from the above steps). An equal amount of DNaseI-treated RNA was used as a negative (no RT) control, whereas the GAPDH gene served as an internal control. The qRT-PCR was performed on Applied Biosystems ViiA 7 system using the 384-well plate and analyzed using QuantStudio^™ Real-Time PCR Software (Applied Biosystems). The initial denaturation of qRT-PCR was at 95°C for 10 min followed by 40 cycles including strand separation at 95°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 30 sec.