Reference genes for RT-qPCR normalisation in different tissues, developmental stages and stress conditions of amaranth

Studies of gene expression are very important for the identification of genes that participate in different biological processes. Currently, reverse transcription quantitative real-time PCR (RT-qPCR) is a high-throughput, sensitive and widely used method for gene expression analysis. Nevertheless, RT-qPCR requires precise normalisation of data to avoid the misinterpretation of experimental data. In this sense, the selection of reference genes is critical for gene expression analysis. At this time, several studies focus on the selection of reference genes in several species. However, the identification and validation of reference genes for the normalisation of RT-qPCR have not been described in amaranth. A set of seven housekeeping genes were analysed using RT-qPCR, to determine the most stable reference genes in amaranth for normalisation of gene expression analysis. Transcript stability and gene expression level of candidate reference genes were analysed in different tissues, at different developmental stages and under different types of stress. The data were compared using the geNorm, NormFinder and Bestkeeper statistical methods. The reference genes optimum for normalisation of data varied with respect to treatment. The results indicate that AhyMDH, AhyGAPDH, AhyEF-1 and AhyACT would be optimum for accurate normalisation of experimental data, when all treatment are analysed in the same experiment. This study presents the most stable reference genes for normalisation of gene expression analysis in amaranth, which will contribute significantly to future gene studies of this species.