Sybr green qpcr master mix 2

RNA was extracted from aerial tissue collected from five individuals of three new independent experiments with the Direct-zol RNA miniprep kit (Zymo Research, R2050) and digested with the included DNAse I in a column. Total RNA (2.1 μg) was used to synthesize cDNA using the Maxima First Strand cDNA kit (ThermoScientific, K1642). Quantitative PCR was performed in a Piko Real 96 thermocycler (ThermoScientific) using SYBR Green qPCR Master Mix 2× (ThermoScientific, K0251) and 1 μL of 1:10 cDNA dilution (20 μL final volume). Primers were designed using on-line tools at www.idt.com (IDT), with sequences downloaded from phytozome.jgi.doe.gov and synthesized by Macrogen. The efficiency was determined in 1:4, 1:6: 1:64 and 1:256 cDNA dilutions using the integrated thermocycler software (Supplementary Table S6).

The bacterial load and abundances of Firmicutes, Bacteroidetes, and Actinobacteria were estimated by qPCR. We randomly selected seven CO, five OB, and seven OMS samples by proportionate stratified sampling method, corresponding to 25% of each group. Each PCR reaction had 12.5 μL of SYBR Green qPCR Master Mix (2×) (Thermo Fisher, Waltham, MA, USA), 0.8 μL of each primer (0.3 μM concentration), 2.5 DNA quenching (equilibrated to 5 ng), and 8.4 μL of water, obtaining a final volume of 25 μL. Each reaction was performed in triplicate. The amplification conditions were the following: a thermal cycling of 5 min at 95 °C denaturation, 30 cycles of 95 °C for 15 s, 61.5 °C for 15 s, and 72 °C for 20 s for alignment and for 5 min at 72 °C for elongation. We used StepOne Real-Time PCR System (Thermo Scientific), primers (Firmicutes, Bacteroidetes, Actinobacteria, and Universal), and conditions developed for each bacterial taxon [52 (link)]. For each pair of primers, we determined the specificity of products and the absence of primer dimers using a melting curve analysis. The Ct was measured by triplicate for each pair of primers. We used an ANOVA test to detect significant differences in the Ct among the groups (Table S5), and a Tukey test to identify specific significant differences between pairs of groups.