Genejet plant rna purification kit

Total RNA was extracted from roots using the GeneJet Plant RNA purification kit (ThermoFischer, www.thermofischer.com). First-strand complimentary DNA (cDNA) was synthesized from 2 μg of total RNA using the RevertAid Reverse Transcriptase (ThermoFischer, www.thermofischer.com). Primer design was carried out using the Primer3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). Primer combinations showing a minimum amplification efficiency of 90% were used in real-time RT-PCR experiments (Supplementary Table 1). For transcript normalization, we used the well-established At1g13320 reference gene encoding a protein phosphatase 2A subunit^{64 (link)}. Real-time RT-PCR amplifications were performed using the Light Cycler Fast Start DNA Master SYBR Green I kit on a Light Cycler apparatus according to manufacturer’s instructions (Roche, www.roche.com). Cycling conditions were as follows: 95 °C for 10 min, 40 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s. PCR amplification specificity was checked using a dissociation curve (55–95 °C). A negative control without cDNA template was included for each primer combination. Experiments included three technical replicates and three independent biological replicates. Ratios were done with constitutive controls for gene expression to normalize the data between different biological conditions.

Total RNA was extracted from 25-day-old wheat seedlings using the GeneJET Plant RNA Purification Kit (Thermo Scientific^TM). Approximately 2 μg of total DNase-treated-RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit by Invitrogen (Karlsruhe, Germany). Expression analysis of ET and polyamine biosynthesis genes in wheat was performed by using primers (Table 1), designed through primer 3.0 (Untergasser et al., 2012 (link)). TaACTIN2 (AB181991) served as an internal control, previously reported by Xu et al. (2013) (link). Amplification of each gene was performed in triplicate by using an ABI PRISM 7900HT sequence detection system (Applied Biosystems), and amplification products were visualized using SYBR Green (Applied Biosystems). Amplification curves were analyzed with a normalized reporter (Rn: the ratio of the fluorescence emission intensity of SYBR Green to the fluorescence signal of the passive reference dye). RT-qPCR expression analysis was performed by using three independent biological replicates with at least three technical replicates.

Murashige and Skoog medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, 2-(N-morpholino)ethanesulfonic acid (MES), triphenil-tetrazolium chloride (TTC), xylenol orange, EDTA, succinate, 4-morpholinepropanesulfonic acid (MOPS), Polyvinylpyrrolidone (PVP-40), hydroxylamine, sulphanilamide, α-naphthylamine, ampicillin, NP40, safranin, Salicylhydroxamic acid (SHAM), kalium-cyanide (KCN), linoleic acid, fatty acid free bovine serum albumin (BSA), luminol, p-Coumaric acid, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), were obtained from Sigma-Aldrich. ProBond Purification System was purchased from Invitrogen. Amicon Ultra 30K Centrifugal Filter Units were purchased from Merck. IPTG was obtained from Duchefa Biochemie, cytochrome c was purchased from Fluka. Primary and secondary antibodies were purchased from Agrisera Antibodies. All other chemicals were of analytical or HPLC grade, and were purchased from Reanal, Hungary. Pierce BCA Protein Assay Kit, GeneJET Plant RNA Purification Kit, and RevertAid First-Strand cDNA Synthesis Kit were obtained from Thermo Scientific; SensiFAST SYBR No-ROX Kit was purchased from Bioline.

Total RNA from leaf tissue was extracted using a GeneJET^™ Plant RNA Purification Kit (Thermo Scientific^™) following the manufacturer’s instructions. DNase treatment of RNA was performed using DNase 1 solution (ThermoFisher^™). The concentration of RNA was measured using a NanoDrop^™ 1000 (Thermo Scientific^™) and the integrity was visualized on a agarose gel after electrophoresis [23 (link)]. Reverse transcription of total RNA was performed as described previously [28 (link)] using a cDNA Reverse Transcription Kit (Applied Biosystems). The reaction mixture (15 μL containing 2 ng of cDNA synthesized with 300 nM of gene specific primers (Invitrogen^™) (Table 2) and 7.5 μL SYBR Green master mix (BioRad) was loaded onto qPCR reaction plates (Applied Biosystems). The reactions were run on a StepOnePlus Real-Time PCR system according to Applied Biosystems protocols. Using the comparative C_T method (ΔΔC_T) method with GAPDH as a control, the relative expression of four genes of interest (TaPR1, TaPR2, TaPR3 (Chitinase) and TaGlu2) were calculated [28 (link),29 (link)].

Total RNA was isolated from 100 mg of fresh leaf tissue harvested at 5 dpi by using the GeneJET Plant RNA Purification Kit (Thermo Fisher Scientific). The RNA was treated with rDNase (Invitrogen) before cDNA synthesis. The cDNA was synthesized from 1 µg of total RNA using the PrimeScript™ RT-PCR Kit (Takara). Three technical replicates were measured for gene expression levels in each cDNA sample. The SYBR® fluorescent dye (TB Green® Premix Ex Taq™, Takara) was employed for reporting the amplification of cDNA in the Applied biosystem 7500 Fast Real-Time PCR system with specific primer pairs for either DFR or PAL2 genes (Additional file 1: Table S2). F-BOX gene was amplified as an internal reference (Liu et al., 2012) for determining DFR/PAL2 gene expression in each sample (ΔCt). The differential DFR/PAL2 gene expression levels were calculated according to the comparative ΔΔCt method as the ratio between the samples agroinfiltrated with the dCasEV2.1 and a gRNA targeting the DFR/PAL2 promoter (gNbDFR/gNbPAL2) and those including the same construct but an unrelated gRNA (gMTB), both for copper and water treatments [19 ].

A sample for RNA extraction was obtained by taking shoot tips along with several leaves, with a combined weight of up to 100 mg. The samples were processed with a GeneJET Plant RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the extracted RNA was quantified and quality-controlled using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific).

Three-week-old plants were subjected to dehydration on bench for 5 and 8 h before the whole treated plants were collected and frozen in liquid nitrogen. Purified total RNA was obtained from these samples by using GeneJET Plant RNA Purification Kit (Thermo Scientific, Waltham, MA, USA) and RapidOut DNA Removal Kit (Thermo Scientific, Waltham, MA, USA). For cDNA synthesis, 1 µg of total RNA and Maxima First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) were used. The procedure for RT-qPCR was conducted according to previously described study [67 (link)], with Actin2 being used as internal reference gene [104 (link)]. Sequences of specific primers for reference and stress-related target genes are listed in Supplementary Table S1. Comparative expression analysis was performed by using the 2^−ΔΔCt method [105 (link)]. Three biological replicates from each genotype were used in each treatment condition in this assay.