Nzyspeedy qpcr green master mix

Total genomic DNA was extracted after 48 h of exposure to the different scenarios established (0.25 g of soil; three replicates per condition) using a PureLink microbiome DNA purification kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA), following manufacturer’s instructions.
Quantitative PCR (qPCR) was used to determine total bacterial abundance. The quantification of the 16S rRNA gene was carried from each DNA sample in three independent DNA reactions with primers 338F (CCTACGGGAGGCAGCAG) and 518R (CCTACGGGAGGCAGCAG) [29 (link)]. The amplification reaction mixture (final volume of 20 μL) contained 2 μL of the DNA template, 0.4 μL of each primer solution at 10 µM, 10 µL of NZYSpeedy qPCR Green Master Mix (3 mM MgCl₂) (Nzytech, Portugal) and 7.2 μL of ultra-pure water. PCR reactions were performed using a CFX96™ real-time PCR system (Bio-Rad, Hercules, CA, USA) under the following thermal conditions: 95 °C for 3 min, followed by 30 amplification cycles consisting of 95 °C for 15 s and 65 °C for 30 s. Melting analysis was performed from 55 °C to 95 °C, with steady 0.1 °C increments at each 5 s. Quantification was performed based on the standard curve method, as described by Brankatschk et al. [30 (link)].

ChIP analysis was performed in fully expanded leaves from 4-week-old pretreated plants (50 μMAA or Mock + 0.02% tween 20 by spray). Three dat, fully expanded leaves from at least 12 different plants/treatment were included in each of the samples (following a randomized block design). Each condition was constituted by two-four different samples (two replicates were used only in BTH treatments as internal experimental control). Chromatin isolation and analysis were conducted as described in Haring et al. (2007 (link)) from 2 g of leaf tissue per sample. Chromatin immunoprecipitation was performed, using EpiQuik Chromatin Immunoprecipitation Kit (P-2002, Epigentek) with the antibody antiH3K4m3 (#07-473 Millipore). Immunoprecipitated samples were quantified by q-PCR analysis in a 7,500 real-time PCR system (Applied Biosystems), using NZYSpeedy qPCR Green Master Mix (MB22303, Nzytech) and specific primers previously reported by Jaskiewicz et al. (2011 (link)). Relative levels were calculated by the method of the reference sample, as described in Rao et al. (2013 (link)). The total amount of DNA/sample was corrected, using values of input aliquots (non-immunoprecipitated) of each sample. Finally, the values were expressed as relative rates, being “1” the average of the control, Mock. ChIP analyses were performed three times with similar results.

RNA extractions from 1 × 10E6 cells were performed with Quick RNA Miniprep Kit (Zymo Research). 140 ng-500 ng RNA were retro-transcribed with Reverse Aid reverse transcriptase (Thermo Scientific). Quantitative PCR (qPCR) was performed with NZY Speedy qPCR Green Master mix (NZY tech) and in a LightCycler 480 Real-Time PCR System (Roche). Primer sequences are detailed in the Supplementary Table S10. Quantifications were normalized to an endogenous control (Glyceraldehyde 3-phosphate dehydrogenase, GAPDH). The relative quantification value for each target gene compared with the calibrator is expressed as 2^(-ΔΔCt).

Quantitative PCR gene expression analysis of 16 genes coding for ATP synthase (atpA, atpB, atpF, atpI), NADH dehydrogenase (ndhA, ndhF, ndhH), cytochrome b/f complex (petB, petD), photosystem I and II (psaA, psaB, psbA, psbB, psbC, psbD), and RuBisCO large subunit (rbcL) proteins was undertaken using NZYSpeedy qPCR Green Master Mix (NZYTech) following the instructions provided, with a 50-fold diluted cDNA template. The reactions were performed in a 96-well plate, with two technical replicates in a CFX96 Touch System (Bio-Rad, Hercules, CA, USA). All the primers (Table S1) were designed from an A. unedo chloroplast (JQ067650.2) and 18S (AF206853.1) sequences, using Primer-BLAST (available at: http://www.ncbi.nlm.nih.gov/tools/primer-blast (accessed on 19 November 2023)). The gene expression was normalized for the 18S reference gene, and the relative expression was calculated according to the Pfaffl method [39 (link)].

For the transcriptional analysis of cell wall biosynthetic and regulatory genes, cells from the pdr18Δ deletion mutant strain were harvested during cultivation as described above for other assays. Exponentially growing cells from the parental strain cultivated in unstressed conditions were also harvested, to be used as a reference value in transcript quantification. Total RNA extraction was performed by the hot phenol method [53 (link)]. The quantitative real-time Reverse Transcription–PCR (qRT–PCR) protocol was used to determine the mRNA levels from RLM1, FKS1, FKS2, BGL2, CHS3, CRH1, GAS1, and PRM5 genes following the manufacturer’s instructions. Primers were designed using Primer Express software V3.0 (Applied Biosystems) and are listed in Supplementary Table S1. The RT–PCR reaction was conducted in a thermal cycler block (Cleaver GTC965), and the qPCR was conducted in QuantStudio 5 (Applied Biosystems) using NZYSpeedy qPCR Green Master Mix (NZYTech). The ACT1 mRNA level was used as the internal control. The value obtained for each target gene at the initial time-point (0 h) for the parental strain cells cultivated under unstressed conditions was set as 1, and the remaining values are relative to this reference value. The results were obtained from at least three independent experiments, and statistically significant differences were tested using one-way ANOVA.

Total RNA was extracted from the left ventricular tissue of young and old mice and from MAECs using the miRNeasy Micro Kit (Qiagen, 217,084) or the RNeasy Plus Micro kit (QIAGEN, 74,034), respectively. Real-time quantitative PCR (RT-qPCR) of 5 ng RNA used for cDNA synthesis was performed using NZYSpeedy qPCR Green Master Mix (NZYtech, MB224) according to the manufacturer’s instructions, and detection was carried out in the CFX Connect Real-Time PCR system (Bio-Rad).

Cells were crosslinked with formaldehyde (37%) and chromatin was immunoprecipitated using the iDeal Chip-seq Kit for Transcription Factors (Diagenode, Belgium), according to the recommended protocol. Rabbit monoclonal antibody specific for c-Myc (Abcam®) was used to immunoprecipitate chromatin fragments and rabbit IgG antibody was used as negative control. Real-time PCR was performed using NZYSpeedy qPCR Green Master Mix (NZYTech, Portugal). Sequences of primers used to amplify ChIP samples were: primer forward 5′-TGCTTGGCCTGAAATTCTTAG-3′ and primer reverse 5′-ACCAGGGCAAGATACAGGA-3′. To analyze the results, the percentage input method was used.