PMID- 38221964 OWN - NLM STAT- PubMed-not-MEDLINE LR - 20240116 IS - 2471-1411 (Electronic) IS - 2096-1790 (Print) IS - 2471-1411 (Linking) VI - 8 IP - 3 DP - 2023 Sep TI - miR-26a/30d/152 are reliable reference genes for miRNA quantification in skin wound age estimation. PG - 230-240 LID - 10.1093/fsr/owad037 [doi] AB - 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. CI - (c) The Author(s) 2023. Published by OUP on behalf of the Academy of Forensic Science. FAU - Suo, Longlong AU - Suo L AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Cheng, Jian AU - Cheng J AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Yuan, Haomiao AU - Yuan H AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Jiang, Zhenfei AU - Jiang Z AD - Department of Road Traffic Accident Investigation, Academy of Forensic Science, Shanghai, China. FAU - Tash, Dilichati AU - Tash D AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. AD - Autonomous Prefecture Public Security Bureau, Xinjiang Uygur Autonomous Region, Urumqi, China. FAU - Wang, Linlin AU - Wang L AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. AD - Collaborative Laboratory of Intelligentized Forensic Science, Shenyang, China. AD - Laboratory of Forensic Biochemistry, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Cheng, Hao AU - Cheng H AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Zhang, Zhongduo AU - Zhang Z AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Zhang, Fuyuan AU - Zhang F AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Zhang, Miao AU - Zhang M AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. AD - Collaborative Laboratory of Intelligentized Forensic Science, Shenyang, China. AD - Laboratory of Forensic Biochemistry, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Cao, Zhipeng AU - Cao Z AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. AD - Collaborative Laboratory of Intelligentized Forensic Science, Shenyang, China. AD - Laboratory of Forensic Biochemistry, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Zhao, Rui AU - Zhao R AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. AD - Collaborative Laboratory of Intelligentized Forensic Science, Shenyang, China. AD - Laboratory of Forensic Biochemistry, China Medical University School of Forensic Medicine, Shenyang, China. FAU - Guan, Dawei AU - Guan D AD - Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China. AD - Collaborative Laboratory of Intelligentized Forensic Science, Shenyang, China. AD - Laboratory of Forensic Biochemistry, China Medical University School of Forensic Medicine, Shenyang, China. LA - eng PT - Journal Article DEP - 20231015 PL - England TA - Forensic Sci Res JT - Forensic sciences research JID - 101724928 PMC - PMC10785593 OTO - NOTNLM OT - forensic genetics OT - forensic sciences OT - miRNA OT - normalization OT - qRT-PCR OT - reference gene OT - skin wound healing OT - wound age estimation EDAT- 2024/01/15 06:42 MHDA- 2024/01/15 06:43 PMCR- 2023/10/15 CRDT- 2024/01/15 04:26 PHST- 2021/06/05 00:00 [received] PHST- 2023/08/23 00:00 [accepted] PHST- 2024/01/15 06:43 [medline] PHST- 2024/01/15 06:42 [pubmed] PHST- 2024/01/15 04:26 [entrez] PHST- 2023/10/15 00:00 [pmc-release] AID - owad037 [pii] AID - 10.1093/fsr/owad037 [doi] PST - epublish SO - Forensic Sci Res. 2023 Oct 15;8(3):230-240. doi: 10.1093/fsr/owad037. eCollection 2023 Sep.