Proflex flat pcr system

For the highest levels of sensitivity, a quantitative PCR approach of digital PCR was performed with a QuantStudio 3D Digital PCR System (Thermo Fisher Scientific, USA) to validate the Real-Time PCR data on selected samples and obtain further quantitative information. Quantitative TaqMan assays were developed based on 16S rRNA sequencing results in the V2 region of the bacterial sequences obtained by Ion Reporter cloud software. Cercospora quantification was also done using dPCR (Supplementary Figs. S4, S5). The PCR mix contained 8 μl of QuantStudio 3D Digital PCR Master Mix v2, 1.44 μl (10 μM) of forward and reverse primers, 0.40 μl of FAM probe, and 3.22 μl of nuclease-free water. Chips were loaded with 14.5 μl of mix and 1.5 μl of DNA at a concentration of 10 ng μl^-1. Amplification was performed on a ProFlex Flat PCR system (Thermo Fisher Scientific, USA) with the following thermal profile: 96 °C for 10 min, 40 cycles at 60 °C for 2 min, 98 °C for 30 s, and a final step at 60 °C for 2 min. Data were analyzed with a QuantStudio 3D Digital PCR Analysis Suite Software v3.0 (Thermo Fisher Scientific, USA).

Total RNA was extracted from the heart tissues using TRIzol (Invitrogen, Waltham, MA) and then transcribed to cDNA by using superscript III reverse transcriptase and random primers following the manufacturer's instruction (Invitrogen). Target genes expression levels were determined through SYBR Green PCR Kit using ProFlex Flat PCR System (ThermoFisher). The primers used were as follows (5’-3’):