Selection of suitable reference genes for miRNA expression normalization by qRT-PCR during flower development and different genotypes of Prunus mume

• The choice of suitable reference genes is crucial in miRNA gene expression studies.
• We examined the expression of four protein-coding genes and four ncRNA genes.
• Reference gene expression was analyzed with the two programs geNorm and NormFinder.
• Expression of two miRNA genes was examined during floral development in P. mume.
• Protein-coding genes PP2A-2 and UBC are stable reference genes for miRNA studies during floral development in P. mume.

Prunus mume is widely cultivated in East Asia owing to its favored ornamental characteristics. Reverse-transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for miRNA expression analysis that requires carefully selected reference genes to ensure data reliability. This study aimed to identify and evaluate reference genes for miRNA expression using qRT-PCR in P. mume. Four protein-coding genes (PP2A-1, PP2A-2, UBC and ACT) and four ncRNAs (U5, U6, 5S and 5.8S) were chosen, and their expression levels were assessed by qRT-PCR in three sample sets: (1) bud development and flowering in P. mume, (2) fully open flowers of different genotypes in P. mume and (3) all P. mume samples. The stability and suitability of the candidate reference genes were validated using the commercially available programs geNorm and NormFinder. We found that PP2A-2 and UBC were suitable reference genes for bud development and flowering and in the different genotypes of P. mume. However, 5.8S was an unsuitable reference gene in these studies. Interestingly, the expression of most protein-coding genes was more stable than that of the ncRNAs in the experimental samples. Finally, the expression of the Pmu-miR156a and Pmu-miR172a genes was assessed to allow comparisons between selected candidate reference genes, highlighting the importance of careful reference gene selection.