miR-26a/30d/152 are reliable reference genes for miRNA quantification in skin wound age estimation.

Unlabelled: MicroRNAs (miRNAs) are a class of small non-coding RNAs that exert their biological functions as negative regulators of gene expression. They are involved in the skin wound healing process with a dynamic expression pattern and can therefore potentially serve as biomarkers for skin wound age estimation. However, no reports have described any miRNAs as suitable reference genes (RGs) for miRNA quantification in wounded skin or samples with post-mortem changes. Here, we aimed to identify specific miRNAs as RGs for miRNA quantification to support further studies of skin wound age estimation. Overall, nine miRNAs stably expressed in mouse skin at certain posttraumatic intervals (PTIs) were preselected by next-generation sequencing as candidate RGs. These nine miRNAs and the commonly used reference genes (comRGs: U6, GAPDH, ACTB, 18S, 5S, LC-Ogdh) were quantitatively examined using quantitative real-time reverse-transcription polymerase chain reaction at different PTIs during skin wound healing in mice. The stabilities of these genes were evaluated using four independent algorithms: GeNorm, NormFinder, BestKeeper, and comparative Delta Ct. Stability was further evaluated in mice with different post-mortem intervals (PMIs). Overall, mmu-miR-26a-5p, mmu-miR-30d-5p, and mmu-miR-152-3p were identified as the most stable genes at both different PTIs and PMIs. These three miRNA RGs were additionally validated and compared with the comRGs in human samples. After assessing using one, two, or three miRNAs in combination for stability at different PTIs, PMIs, or in human samples, the set of miR-26a/30d/152 was approved as the best normalizer. In conclusion, our data suggest that the combination of miR-26a/30d/152 is recommended as the normalization strategy for miRNA qRT-PCR quantification in skin wound age estimation.

Key Points: The small size of miRNAs makes them less susceptible to post-mortem autolysis or putrefaction, leading to their potential use in wound age estimation.Studying miRNAs as biological indicators of skin wound age estimation requires the selection and validation of stable reference genes because commonly used reference genes, such as U6, ACTB, GAPDH, 5S, 18S, and LC-Ogdh, are not stable.miR-26a/30d/152 are stable and reliable as reference genes and their use in combination is a recommended normalization strategy for miRNA quantitative analysis in wounded skin.