Computational protocol: Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem

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Protocol publication

[…] Expression data generated in this work, along with those generated in previous RNA-seq experiments shown in , were used for the selection of the most stably expressed genes across all the treatments. Nine genes with lower variation coefficient (VC), calculated as the ratio between the standard deviation and the average of each gene expression (RPKM, reads per kilobase of transcript per million mapped reads) across all the treatments and biological replicates, were selected (). Additionally, two traditional reference genes used in tomato (GADPH and EF1α) and PHD, the most stably expressed tomato gene identified in a previous report using Xcv infected tomato plants were included for analysis.The nucleotide sequence of each gene was downloaded from the Sol Genomics webpage and primers were designed using the PrimerQuest tool (Integrated DNA Technologies). Primer efficiencies were checked by RT-qPCR using different cDNA dilutions (). Dissociation curves were performed to show amplification specificity (). [...] Data obtained from the RT-qPCR experiments were analyzed using three statistical programs: geNorm, NormFinder and BestKeeper.Expression of one PTI- (Solyc02g069960) and one ETI-specific gene (Solyc09g092500) was analyzed by RT-qPCR as explained above. The data obtained was normalized using the two best and the worst reference genes and the relative expression was expressed as E−ΔΔCq, where E corresponds to the primer efficiency value. […]

Pipeline specifications

Software tools PrimerQuest, NormFinder, BestKeeper
Applications RNA-seq analysis, qPCR
Organisms Solanum lycopersicum, Pseudomonas syringae, Bacteria