Selection of suitable inner reference genes for relative quantification expression of microRNA in wheat

Quantitative real-time PCR (qRT-PCR) is currently the most accurate and widely applied method to detect differential genes expression, but choosing a suitable gene to be the internal control is a crucial factor for correct analysis of the results. MicroRNAs are fundamental regulatory genes of eukaryotic genomes, acting on several biological functions. Transcription accumulation of microRNAs has been studied using qRT-PCR, while no validated reference genes for microRNAs in wheat are available until now. In this study, nine previous reported housekeeping genes and ten wheat microRNAs were examined with regard to their use as normalizer and data was analyzed using geNorm and NormFind software. Expression stability of candidate inner reference genes was investigated in different conditions. After analysis of all the sample pools and samples after biotic and abiotic stress treatments, it was found that microRNAs had better expression stability than protein-coding genes, and mi167 and mi159 appeared to be the two most suitable reference genes in wheat. To confirm the stable expression of the putative reference genes in wheat, expression of mi171b of wheat was examined with inner reference genes mi167, mi159 and combination of mi157 and mi159 respectively. We provided evidence for that in order to get a more accurate result of gene expression, mi167 and mi159 should be used as inner reference gene for normalization together.

► Both protein coding genes and miRNAs were examined when they were as normalizers.
► The stable expression of the putative reference genes was confirmed in wheat.
► Direct evidence was provided that miRNA can be used as inner reference gene.