Differential thermal stability of mRNA, miRNA and lncRNA in purified human tissue-derived RNA extracts: a proof-of-principle study with forensic implications.

RNA-based markers hold considerable potential for forensic transcriptomics applications, yet their practical use is constrained by heterogeneous RNA degradation under environmental stress conditions. The thermal stability profiles of different RNA classes in degraded biological samples remain incompletely characterized. Here, we established an in vitro thermal degradation model using commercially purified total RNA extracts derived from human brain, liver, and kidney tissues, and performed whole-transcriptome sequencing to systematically compare the degradation patterns of mRNAs, lncRNAs, and miRNAs under extreme thermal stress. RNA Integrity Number (RIN) values decreased progressively with increasing treatment duration. Notably, high-quality sequencing data were still obtainable from RNA extracts with RIN values as low as approximately 3.5. Degradation kinetics exhibited significant differences across both RNA sources and RNA classes. In brain-derived RNA extracts, lower decay rate constants (indicating greater stability) were associated with shorter transcript lengths and shorter 5' untranslated regions (UTRs) for mRNAs, while stable miRNAs showed higher GC content and lower minimum free energy. In liver-derived RNA extracts, lower decay rate constants were correlated with longer transcript lengths and longer 3' UTRs, whereas no consistent structural associations with stability were identified in kidney-derived RNA extracts. These observations suggest that thermal RNA degradation may differ according to RNA source and RNA class, a possibility that warrants further validation in larger studies using degraded forensic specimens.