M mulv cdna synthesis kit

Total RNA was isolated from 0, 75 and 150 mM NaCl treated non-transformed BY-2 cells after pretreatment with or without 2 µM of plant PeptoQ for 2 h (samples were collected at 1 and 3 h from three independent biological repeats) using the InnuPREP plant RNA kit (Analytik Jena AG, Jena, Germany) according to the manufacturer’s instructions. The cDNA synthesis was performed from 1 µg of total RNA extracts using the M-MuLV cDNA Synthesis Kit (NEB), with Oligo(dTs) (Thermo Fisher, Germany) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was carried out on a CFX Touch real-time PCR system (Bio-Rad, Munich, Germany) detection system according to the manufacturer’s instructions with the following cycler conditions: 3 min, 95 °C, 39 × (95 °C for 15 s, annealing at 60 °C for 40 s). Details on the primer sequences and accession numbers for genes of interest and reference genes are listed in Suppl. Table S1. To compare the transcript levels between different samples, the 2^−∆∆Ct method was used, the difference in the cycle threshold (Ct) values between the endogenous control genes (elongation factor 1 α (GenBank D63396) and ribosomal protein L25 (GenBank L18908) and target gene was calculated^{89 (link)}. Data are given relative to the values obtained in control cells that had neither been treated with the peptoid, nor with salt.

Total RNA was isolated using the Universal RNA Purification Kit (Roboklon, Germany). We used the optional on-column DNase (Qiagen, Hilden, Germany) digestion was conducted as defined in the protocol of the producer. Quantity and quality of the obtained RNA were checked spectrophotometrically (NanoDrop, Radnor, USA), and, in parallel, by electrophoresis on a 0.8% agarose gel. For reverse transcription of the mRNA into cDNA using the M-MuLV cDNA Synthesis Kit (New England Biolabs; Frankfurt am Main, Germany) followed the instructions of the manufacturer. The amount of RNA template was adjusted to 1 μg.
After quantitative RT-PCR (qPCR) using a CFX96^TM real-time PCR cycler (Bio-RAD, USA) expression levels of target genes were calculated with the 2^-ΔΔCt method^{71 (link)} and normalised to elongation factor 1 (EF1-α) as a house-keeping gene. Genes involved in grapevine basal immunity such as the phenylpropane phytoalexin synthesis genes phenylalanine ammonia lyase (PAL), stilbene synthase (StSy), and resveratrol synthase (RS), the transcription factor MYB14 as regulator of stilbene synthesis, and the jasmonate ZIM/tify-domain protein 1 (JAZ1) as a readout for jasmonate signalling were used. Accession numbers of these genes and the primer details are given in Table S1. Each experiment represents three biological replicates, each done as technical triplicate.

Rice (Oryza sativa L. japonica cultivar Nipponbare) seedlings were grown in darkness at 25 °C for 4 days and coleoptiles excised and shockfrozen in liquid nitrogen in aliquots of 100 mg, followed by grinding in a TissueLyser (Qiagen/Retsch Hilden, Germany). Total RNA was extracted with the innuPREP Plant RNA kit (Analytik, Jena, Germany), including on-column digest of genomic DNA with RNase-free DNAse I (Qiagen) according to the manufacturer instructions. After quality check by electrophoresis on 1% [w/v] agarose gels, 1 μg RNA was used for cDNA synthesis with the M-Mulv cDNA Synthesis Kit (NEB). Plasmids for plant transformation were constructed via GATEWAY^® cloning as described in Klotz and Nick^{11 (link)}. The full-length coding sequence of OsDLK was amplified from the cDNA template with a pair of primers containing attB-sites. To get the full-length OsDLK the forward primer: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCCACGCGCGCCACTCGCC-3′, and the reverse primer: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCCTTGCGCCAAGCTACGCACT-3′ were used.