Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues

Abstract

Gene expression profiling via quantitative real-time PCR is a robust technique widely used in the life sciences to compare gene expression patterns in, e.g., different tissues, growth conditions, or after specific treatments. In the field of plant science, real-time PCR is the gold standard to study the dynamics of gene expression and is used to validate the results generated with high throughput techniques, e.g., RNA-Seq. An accurate relative quantification of gene expression relies on the identification of appropriate reference genes, that need to be determined for each experimental set-up used and plant tissue studied. Here, we identify suitable reference genes for expression profiling in stems of textile hemp (Cannabis sativa L.), whose tissues (isolated bast fibres and core) are characterized by remarkable differences in cell wall composition. We additionally validate the reference genes by analysing the expression of putative candidates involved in the non-oxidative phase of the pentose phosphate pathway and in the first step of the shikimate pathway. The goal is to describe the possible regulation pattern of some genes involved in the provision of the precursors needed for lignin biosynthesis in the different hemp stem tissues. The results here shown are useful to design future studies focused on gene expression analyses in hemp.

Cell wall composition and transcriptomics in stem tissues of stinging nettle (Urtica dioica L.): Spotlight on a neglected fibre crop

“…Samples were reduced to a fine powder using a mortar, a pestle and liquid nitrogen. Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer's instructions (with the on-column DNase digestion), as described in and Mangeot-Peter, Legay, Hausman, Esposito, and Guerriero (2016). RNA quantity and quality were assessed with a NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and a 2100 Bioanalyzer (Agilent).…”

Sucrose synthase gene expression analysis in the fibre nettle (Urtica dioica L.) cultivar “clone 13”

“…After 5 weeks of growth, samples were taken along three regions of the stem localized at different heights (i.e. top, middle and bottom), following the same sampling strategy described for textile hemp by our group (Guerriero et al, 2017;Mangeot-Peter et al, 2016). The "TOP" segment is the internode located at the top (below the apex).…”

“…A segment of 2.5 cm was collected from the central portion of each internode, to avoid variations in gene expression due to the differences in developmental stages of the cell types (as described in textile hemp by Guerriero et al, 2017). Each internode was peeled to separate the cortex from the core, as previously described in hemp (Mangeot-Peter et al, 2016). All collected samples were frozen in liquid nitrogen and stored at -80°C until analysis.…”

“…Nine candidate reference genes (i.e. tubulin, actin, the translation elongation factor EF2, the eukaryotic translation initiation factor eTIF4E, cyclophilin, the tonoplast intrinsic protein TIP41, histone3, the gene coding for small GTP-binding protein RAN, the gene corresponding to the calcium-dependent protein kinase CDPK) chosen from the list of genes reported in textile hemp (Mangeot-Peter et al, 2016), were validated in this study. The sequences of the SUS and the candidate reference genes are listed in Supplementary file 1.…”

“…The cDNA was amplified using the SYBRGreen qPCR Master Mix. The reactions were run in technical triplicates using the cycling parameters and melt curve analysis described previously (Mangeot-Peter et al, 2016). Gene expression was calculated using qbase + version 3.1 (Biogazelle, Zwijnaarde, Belgium, www.qbaseplus.…”