Quantitect reverse transcription kit

PBS was from Oxoid. DNAse- and RNAse-free water was from VWR. Recombinant human TNF and IL-6 ELISA Duoset were from R&D systems. Quantitect primer assays for TLR1-7 and 18S were from Qiagen. QuantiTect Reverse Transcription kit, RNeasy minikit, Leupeptin, pepstatin and LightCycler 480 SYBR Green I Master mix were from Roche Molecular Biochemicals. RNAlater was from Life technologies. FSL-1 and Pam₃CSK₄ were from Invivogen. [³H]-arachidonic acid ([³H]-AA), and liquid scintillation cocktail Ultima Gold were from NEN Perkin Elmer. AVX002 and Inhibitor 28 were provided by Avexxin AS (Trondheim, Norway). Arachidonyl trifluoromethyl ketone (AACOCF3, ATK) was from Enzo Life Sciences. Varespladib (LY315920) was from Selleckchem. CAY10502, CAY10590 and PGE₂ ELISA kit were from Cayman Chemicals. Phospho-cPLA2 (Ser505) antibody was from Cell Signal Technology. α-tubulin antibody was from Santa Cruz Biotechnology. Polyclonal goat anti-mouse immunoglobulins horse radish peroxidase-conjugated secondary antibody was from Dako. Hybond-C nitrocellulose membranes were from GE healthcare. NuPAGE gel system (10% Bis-Tris gel) was from Invitrogen. Hybond-C nitrocellulose membranes were from GE healthcare. SuperSignal West Femto Maximum Sensivity substrate was from Thermo Scientific. All other reagents were from Sigma-Aldrich.

RT-qPCR was performed by TaqMan (TaqMan Gene Expression Master Mix, Thermo Fisher) or SYBR Green assay (LightCycler 480 SYBR Green I Master, Roche) following cDNA synthesis (Superscript III First Strand Synthesis System or QuantiTect Reverse Transcription Kit, respectively). The following TaqMan probes (Thermo Fisher) were used: Nppa (Mm01255747_g1), Nppb (Mm01255770_g1), Myh7 (Mm00600555_m1), Acta1 (Mm00808218_g1), Hprt (Mm01545399_m1), Hif1a (Mm00468869_m1), Vegfa (Mm01281449_m1), Kit (Mm00445212_m1). For SYBR Green assays Renin (forward: 5′-GAG GCC TTC CTT GAC CCA TC-3′, and reverse: 5′-TGT GAA TCC CAC AAG CAA GG-3′) and Gapdh (forward: 5′-CTT GGG CTA CAC TGA GGA C-3′, and reverse: 5′-CTG TTG CTG TAG CCG TAT TC-3′) primers were used from Integrated DNA Technologies.

Quantitative PCR was performed as previously described (Øverby et al., 2015 (link)). Briefly, fresh plant material was snap-freezed in liquid nitrogen and subsequently grounded from which total RNA was extracted using Spectrum Plant Total RNA Kit. RNase-Free DNase Set was used to prevent DNA contamination. RNA concentration was measured using NanoDrop 1000 (Thermo Scientific). cDNA was synthesized using QuantiTect Reverse Transcription Kit, and qPCR was performed with SYBRgreen in 96-well plate in a Lightcycler 480 (Roche Applied Science) with the following program: preincubation step (95°C, 5 min), 45 amplification cycles (95°C, 10 s; 55°C, 10 s; 72°C, 10 s), and a final melting curve analysis. Cycle threshold values, PCR efficiencies and relative expression values were calculated using Lightcycler 480 Software (Roche), LinRegPCR and REST 2009 (QIAGEN), respectively. At4g24550 (Clathrin) and At4g34270 (TIP41-like) were used as housekeeping genes. Primer sequences are given in Supplementary Table S1.