Computational protocol: Distinct transcriptome responses to water limitation in isohydric and anisohydric grapevine cultivars

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Protocol publication

[…] As stated above, leaf samples were taken after 2, 6 and 27 days of WS, and 24 days after re-watering (three pools of leaves, each consisting of four pieces). At the same times, three pools of 15 berries were taken from the bunch opposite the node corresponding to the leaf sample. All samples were immediately frozen in liquid N2 and stored at −80 °C.Total RNA was extracted from ~50 mg of frozen leaves and ~200 mg of berry (pericarp plus seeds) using the Spectrum™ Plant Total RNA kit (Sigma-Aldrich) as previously described []. We hybridized 5 μg of total RNA per sample to a NimbleGen microarray 090818_Vitus_exp_HX12 chip (Roche, NimbleGen Inc., Madison, WI), according to the manufacturer’s instructions []. Statistical analysis of the microarray data was carried out using TMeV v4.8 (mev.tm4.org/). Statistical analysis of microarrays (SAM) was carried out with a false discovery rate (FDR) of 0.01 % and ANOVA was carried out using α = 0.01 and standard Bonferroni correction. Hierarchical cluster analysis was applied using Pearson’s correlation distance, unless stated otherwise. Principal component analysis (PCA) was carried out using SIMCA P+ v13 (Umetrics, USA). Gene Ontology (GO) annotation was applied to gene clusters and organ-specific genes using the BiNGO v2.3 plug-in tool in Cytoscape v2.6 with PlantGOslim categories, as described by Maere et al. []. Overrepresented PlantGOslim categories were identified using a hypergeometric test with a significance threshold of 0.01 for genes modulated in leaves and 0.05 for genes modulated in berries. STEM v1.3.8 was used for clustering, comparing and visualizing gene expression data []. Line plots were drawn using SigmaPlot v13.0. […]

Pipeline specifications

Software tools TM4, BiNGO, SigmaPlot
Application Miscellaneous
Chemicals Oxygen