miR-26a/30d/152 are reliable reference genes for miRNA quantification in skin wound age estimation
Longlong Suo1, Jian Cheng1, Haomiao Yuan1
1Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China.
Abstract:
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.
Insights
MicroRNAs (miRNAs) are promising biomarkers for estimating skin wound age. This study identified three stable miRNA reference genes (miR-26a/30d/152) for accurate miRNA quantification in wounded skin, even after post-mortem changes.
Area of Science:
- Forensic Science
- Molecular Biology
- Biochemistry
Background:
- MicroRNAs (miRNAs) are key gene regulators involved in skin wound healing.
- Their dynamic expression patterns suggest potential as biomarkers for estimating skin wound age.
- However, suitable reference genes for miRNA quantification in wounded skin, especially with post-mortem intervals (PMIs), are lacking.
Purpose of the Study:
- To identify and validate stable miRNA reference genes (RGs) for accurate miRNA quantification in skin wound age estimation.
- To evaluate the stability of candidate RGs across different post-traumatic intervals (PTIs) and PMIs.
- To compare the performance of novel miRNA RGs against commonly used reference genes (comRGs).
Main Methods:
- Nine preselected miRNAs and six comRGs (U6, GAPDH, ACTB, 18S, 5S, LC-Ogdh) were quantified using qRT-PCR in mouse skin across various PTIs.
- Gene stability was assessed using four algorithms (GeNorm, NormFinder, BestKeeper, comparative Delta Ct).
- Stability was further evaluated in samples with different PMIs and validated in human skin samples.
Main Results:
- mmu-miR-26a-5p, mmu-miR-30d-5p, and mmu-miR-152-3p demonstrated the highest stability across different PTIs and PMIs.
- The combination of miR-26a/30d/152 proved to be the most reliable normalization strategy.
- These miRNA RGs outperformed commonly used reference genes in stability.
Conclusions:
- The combination of miR-26a/30d/152 is recommended as the optimal normalization strategy for miRNA qRT-PCR quantification in skin wound age estimation.
- These stable miRNA reference genes are crucial for advancing research in forensic science and wound analysis.
- The findings provide a robust method for analyzing miRNA expression in challenging forensic samples.
