Gene expression was determined using the Plexset® platform from NanoString Technologies Inc. (Seattle, WA, USA), and results were analysed using the nSolver™ 4.0 software (Seattle, WA, USA). Four reference genes and eight target genes were used for the gene expression analysis (Table S1 ).
The eukaryotic small ribosomal subunit 40S (40S), ubiquitin-conjugating enzyme (UBC), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the protein phosphatase 2 (PP2A) genes were used as reference genes. The pathogenesis-related protein family 1 (PR1), APETALA2 ethylene responsive factor 2 (AP2_ERF2), Glucan endo-1,3-β-glucosidase (PR2), thaumatin-like protein TG4 (PR5), NIM-interacting protein 2 (NIMIN2), downy mildew resistance 6 (DMR6), WRKY transcription factor 70 (WRKY70i), and benzyl alcohol dehydrogenase (BAD) were used as target genes. The two 50 bp long probes needed for each gene when using the Nanostring technologies were described previously [8 (link)]) (Table S1 ) except for DMR6 and WRKY70i, for which the capture probes were:
ACGCCCTCACAATTTTGCTTCAGGACCTCCAAGTCTCAGGCCTACAAGTC and TGGAGGAAATATGGACAAAAGGAGATCCTCAATGCCAAATTTCCAAGGTG and the reporter probes were:
CTCAAGGACGGCAAGTGGATGGCCGTCAAACCCCATCCCAATGCCTTTGT and CTACTTTAGGTGCACACACAAGCCTGATCAAGGTTGCCTAGCAACAAAGC, respectively.
All the probes were synthesised by Integrated DNA Technologies Limited (IDT, Singapore). Total RNA was prepared from approximately 100 mg of ground kiwifruit tissue using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, Auckland, New Zealand), following the supplier’s recommendations. Sample purity and RNA concentrations were determined using a Nanophotometer® (Implen, CA, USA). RNA samples were sent at −80 °C to the Grafton Clinical Genomics of the School of Medical Science, University of Auckland, for processing.
The eukaryotic small ribosomal subunit 40S (40S), ubiquitin-conjugating enzyme (UBC), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the protein phosphatase 2 (PP2A) genes were used as reference genes. The pathogenesis-related protein family 1 (PR1), APETALA2 ethylene responsive factor 2 (AP2_ERF2), Glucan endo-1,3-β-glucosidase (PR2), thaumatin-like protein TG4 (PR5), NIM-interacting protein 2 (NIMIN2), downy mildew resistance 6 (DMR6), WRKY transcription factor 70 (WRKY70i), and benzyl alcohol dehydrogenase (BAD) were used as target genes. The two 50 bp long probes needed for each gene when using the Nanostring technologies were described previously [8 (link)]) (
ACGCCCTCACAATTTTGCTTCAGGACCTCCAAGTCTCAGGCCTACAAGTC and TGGAGGAAATATGGACAAAAGGAGATCCTCAATGCCAAATTTCCAAGGTG and the reporter probes were:
CTCAAGGACGGCAAGTGGATGGCCGTCAAACCCCATCCCAATGCCTTTGT and CTACTTTAGGTGCACACACAAGCCTGATCAAGGTTGCCTAGCAACAAAGC, respectively.
All the probes were synthesised by Integrated DNA Technologies Limited (IDT, Singapore). Total RNA was prepared from approximately 100 mg of ground kiwifruit tissue using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, Auckland, New Zealand), following the supplier’s recommendations. Sample purity and RNA concentrations were determined using a Nanophotometer® (Implen, CA, USA). RNA samples were sent at −80 °C to the Grafton Clinical Genomics of the School of Medical Science, University of Auckland, for processing.
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