Selection and validation of reference genes for quantitative expression analysis of miRNAs and mRNAs in Poplar

BACKGROUND: Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is a rapid and sensitive approach to identify miRNA and protein-coding gene expression in plants. However, because of the specially designated reverse transcription and shorter PCR products, very few reference genes have been identified for the quantitative analysis of miRNA expression in plants, and different internal reference genes are needed to normalize the expression of miRNAs and mRNA genes respectively. Therefore, it is particularly important to select the suitable common reference genes for normalization of quantitative PCR of miRNA and mRNA. RESULTS: In this study, a modified reverse transcription PCR protocol was adopted for selecting and validating universal internal reference genes of mRNAs and miRNAs. Eight commonly used reference genes, four stably expressed novel genes in Populus tremula, three small noncoding RNAs and three conserved miRNAs were selected as candidate genes, and the stability of their expression was examined across a set of 38 tissue samples from four developmental stages of poplar clone 84K (Populus alba × Populus glandulosa). The expression stability of these candidate genes was evaluated systematically by four algorithms: geNorm, NormFinder, Bestkeeper and DeltaCt. The results showed that Eukaryotic initiation factor 4A III (EIF4A) and U6-2 were suitable for samples of the callus stage; U6-1 and U6-2 were best for the seedling stage; Protein phosphatase 2A-2 (PP2A-2) and U6-1 were best for the plant stage; and Protein phosphatase 2A-2 (PP2A-2) and Oligouridylate binding protein 1B (UBP) were the best reference genes in the adventitious root (AR) regeneration stage. CONCLUSIONS: The purpose of this study was to identify the most appropriate reference genes for qRT-PCR of miRNAs and mRNAs in different tissues at several developmental stages in poplar. U6-1, EIF4A and PP2A-2 were the three most appropriate reference genes for qRT-PCR normalization of miRNAs and mRNAs during the plant regeneration process, and PP2A-2 and UBP represent the best reference genes in the AR regeneration stage of poplar. This work will benefit future studies of expression and function analysis of miRNAs and their target genes in poplar. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13007-019-0420-1) contains supplementary material, which is available to authorized users.