Lightcycler 480

Total RNA was isolated from whole imbibed seeds or seedlings and further analysed by RT-qPCR. RT-qPCR was performed in a Roche LightCycler 480 and the SensiFast^TM SYBR^® No-ROX Kit (Bioline), using standard PCR conditions according to the manufacturer’s instructions. The cDNA used for RT-qPCR was prepared from 2 µg of total RNA (prepared with the LiCl method) using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Scientific). We used about 20 ng of cDNA per RT-qPCR sample in 10 µl reactions. Parallel reactions were used to normalize the amount of template cDNA (Merchan et al., 2007 (link)); normalized expression was calculated using the mean expression of three to four control transcripts. The specific primers for the four genes used for transcript abundance normalization [Ntubc2, L25, EF-1α (Schmidt and Delaney, 2010 (link)) and NtPsbA] are included in Supplementary Table S1 available at JXB online. Specificity was confirmed by analyses of the RT-qPCR profiles. Reproducibility of RT-qPCR was achieved by running technical duplicates, and by using two independent cDNA preparations. At least two biological replicates were performed per condition. Transcript levels of COP1, HSP26, PsaG, PsbR, POR, HY5, PHYA1, and PHYB1 genes were analysed using the specific primers also listed in Supplementary Table S1.

For determination of mRNA expression levels, total RNA was recovered using TriFast Reagent (Peqlab, Erlangen, Germany) according to the manufacturer's recommendations. The primers (Metabion, Martinsried, Germany) used in these experiments were: Aldose reductase (AR)_fw: 5′-ATC GCA GCC AAG CAC AAT AA-3′; AR_rev: 5′-AGC AAT GCG TTC TGG TGT CA-3′; TonEBP/NFAT5_fw: 5′-AAT CGC CCA AGT CCC TCT AC-3′; TonEBP/NFAT5_rev: 5′-GGT GGT AAA GGA GCT GCA AG -3′; actin_fw: 5′- CCA ACC GCG AGA AGA TGA-3′; actin_rev: 5′- CCA GAG GCG TAC AGG GAT AG -3′. Experiments were performed on a Roche LightCycler 480, using the SensiMix SYBR One-Step Kit (Bioline, Luckenwalde, Germany) according to the manufacturer's recommendations. Relative mRNA expression of the respective genes was calculated by the 2^−ΔΔCT–method (Livak and Schmittgen, 2001 (link)), using β-actin as housekeeping gene. Specificity of PCR productformation was confirmed by monitoring melting point analysisand by agarose gel electrophoresis as described (Küper et al., 2012b (link)).

DNA and RNA isolation, cDNA synthesis, cloning, and PCR amplification was as described by Schullehner et al. (2008) (link). Quantitative reverse transcriptase PCR (qRT-PCR) was carried out with a Roche LightCycler 480 instrument using a Bioline SensiFAST™ SYBR No-ROX kit. Primer and PCR conditions are given in Supplementary Table S1, available at JXB online. DNA sequencing was by Source (Bioscience, Berlin), GATC-Biotech AG (Konstanz, Germany), and Eurofins Genomics (Ebersberg, Germany). All primer pairs were checked for specificity against the maize B73 reference genome, version 2, and PCR products were sequenced to verify amplification of the respective Bx gene.

For determination of NFAT5, S100A4, AR, and β-Actin mRNA expression levels, the total RNA from HK-2 or CaKi-1 cells was prepared by adding TRIFAST Reagent (Peqlab, Erlangen, Germany). The primers (Metabion, Martinsried, Germany) used in this experiment are:
NFAT5_fw: 5′- AAT CGC CCA AGT CCC TCT AC -3′;
NFAT5_rev: 5′- GGT GGT AAA GGA GCT GCA AG -3′;
Actin_fw: 5′- CCA ACC GCG AGA AGA TGA -3′;
Actin_rev: 5′- CCA GAG GCG TAC AGG GAT AG -3′;
S100A4_fw: 5′-CGC TTC TTC TTT CTT GGT TTG-3′;
S100A4_rev: 5′-GAG TAC TTG TGG AAG GTG GAC A-3′;
AR_fw: 5′ATC CGA GCC AAG CAC AAT AA -3′;
AR_rev: 5′-AGC AAT GCG TTC TGG TGT CA -3′
Experiments were caried out on a Roche LightCycler 480 using the SensiMix SYBR One-Step Kit (Bioline, Luckenwalde, Germany) according to the manufacturer's recommendations. Specificity of PCR product formation was confirmed by melting point analysis and by agarose gel electrophoresis.

For determination of NFAT5, AR, HSP70, BGT-1, and β-actin mRNA expression levels, tissues were homogenized in TRIFAST Reagent (Peqlab, Erlangen, Germany), and RNA was isolated as recommended by the manufacturer. Oligonucleotides used in this experiment are listed in Table 1. Experiments were carried out on a Roche LightCycler 480, using the SensiMix SYBR One-Step Kit (Bioline, Luckenwalde, Germany) according to the manufacturer's recommendations. Specificity of PCR product formation was confirmed by monitoring melting point analysis and by agarose gel electrophoresis.

RNA extraction and reverse transcription were performed, as previously described [75 (link)]. RT-PCR was conducted on a Roche Lightcycler 480 machine using SensiFAST™ SYBR Lo-ROX Kit (Bioline, London, UK). Primer sets used for PCR were human/mouse CERT1/Cert1 F-CGATGTGTCCGTGCCAAAAT, R-CCATCCTCCAGGGTTCACATT (designed using Primer-BLAST, National Center for Biotechnology Information, US); mouse Gapdh F-GCCTGGAGAAACCTGCCAAG, R-TCATTGTCATACCAGGAAATG [77 (link)]; human GAPDH F-CGGAGTCAACGGATTTGGTC, R-CCATGGGTGGAATCATATTGG [78 (link)].

RNA was extracted from cells using RNAiso Plus reagent (TaKaRa) and reverse transcribed with gene-specific reverse transcription (RT) primers or random primers using a high-capacity cDNA reverse transcription kit (Applied Biosystems). The real-time PCR was performed using SYBR Premix Ex Taq (Tli RNase H Plus) mix (TaKaRa) in a LightCycler 480 instrument (Roche). Primers used to amplify the alternatively spliced BART RNAs, BARF0, RPMS1, RPMS1A, and A73, are listed in Table 1, and gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The real-time PCR amplification was performed with miRNA-specific TaqMan probes using SensiFAST Probe Lo-ROX mix (Bioline) in a LightCycler 480 instrument. Primers and probes used for BART miRNA detection were described previously (37 (link)). To determine EBV copy number, a 75-bp fragment of the BamH-W region (GenBank accession number V01555.2; nucleotides 17721 to 17796) was amplified by quantitative PCR (qPCR) from phenol-chloroform-extracted DNA. The same fragment was cloned into the TA cloning vector pMD18-T, which was then serially diluted to obtain a standard curve.