Transcriptome-based identification and validation of reference genes for plant-bacteria interaction studies using Nicotiana benthamiana.
Marina A PomboRomina N RamosYi ZhengZhangjun FeiGregory B MartinHernan G RosliPublished in: Scientific reports (2019)
RT-qPCR is a widely used technique for the analysis of gene expression. Accurate estimation of transcript abundance relies strongly on a normalization that requires the use of reference genes that are stably expressed in the conditions analyzed. Initially, they were adopted from those used in Northern blot experiments, but an increasing number of publications highlight the need to find and validate alternative reference genes for the particular system under study. The development of high-throughput sequencing techniques has facilitated the identification of such stably expressed genes. Nicotiana benthamiana has been extensively used as a model in the plant research field. In spite of this, there is scarce information regarding suitable RT-qPCR reference genes for this species. Employing RNA-seq data previously generated from tomato plants, combined with newly generated data from N. benthamiana leaves infiltrated with Pseudomonas fluorescens, we identified and tested a set of 9 candidate reference genes. Using three different algorithms, we found that NbUbe35, NbNQO and NbErpA exhibit less variable gene expression in our pathosystem than previously used genes. Furthermore, the combined use of the first two is sufficient for robust gene expression analysis. We encourage employing these novel reference genes in future RT-qPCR experiments involving N. benthamiana and Pseudomonas spp.
Keyphrases
- genome wide
- genome wide identification
- bioinformatics analysis
- gene expression
- rna seq
- dna methylation
- single cell
- healthcare
- multidrug resistant
- copy number
- machine learning
- transcription factor
- staphylococcus aureus
- high throughput sequencing
- pseudomonas aeruginosa
- wastewater treatment
- big data
- deep learning
- artificial intelligence
- health information
- mass spectrometry
- current status
- essential oil