aly-miR164c-5p: Aly-mir164c-5p is a target miRNA that was investigated in a study using stable and unstable reference genes (RGs) to analyze its expression patterns under various stresses or in different tissues [PMC8304282]. The study found that when unstable RGs were used, the expression of aly-mir164c-5p was significantly different compared to when stable RGs were used [PMC8304282]. Specifically, the expression of aly-mir164c-5p was downregulated under different treatments when stable RGs were used compared to the control [PMC8304282]. The accuracy of stable RG expression was verified using qRT-PCR analysis of three miRNAs, including aly-mir164c-5p, which had relatively high abundance [PMC8304282]. The study also aimed to determine suitable RGs for gene-expression normalization and found that the selected RGs were appropriate for normalizing the expression of three target miRNAs, including aly-mir164c-5p [PMC8304282]. However, when unstable RGs were used, the expression levels of aly-mir164c-5p were often misestimated under various treatments compared to stable RGs [PMC8304282]. Furthermore, it was observed that aly-mir164c-5p and two other miRNAs (aly-miR168a-5p and smo-miR396) had high expression levels in stems or seeds but these levels were often misestimated when unstable RGs were used [PMC8304282]. Overall, this study highlights the importance of using appropriate reference genes for accurate estimation of miRNA expression levels.