Assessing post-mortem interval in cats (Felis catus) through RNA degradation analysis in liver tissue

Estimating time since death, or Post-Mortem Interval (PMI), is a critical component of forensic veterinary pathology. Although many variables are implicated in the decomposition timescale, environmental temperature and its fluctuations significantly affect its progression and the accuracy of PMI estimates. Accumulated Degree Days (ADDs) assist in PMI determination by summing the daily average environmental temperatures. Biomolecular techniques, especially RNA-based analysis, offer promising applications due to the intrinsic instability of RNA after death. In this study, 54 deceased cats collected from veterinary clinics underwent post-mortem (PM) liver sampling. It was performed in 48 cats after exposure to either an outdoor environment ("in field") or stored at + 4 °C ("refrigerated") for 1, 3, 14 and 28 days (6 cats each time point). The remaining 6 cats were sampled within 1 h of death ("controls"). ADDs were subsequently calculated. Microfluidic electrophoresis was performed on RNA extracts from liver samples, obtaining RNA Integrity Number (RIN) and DV₂₀₀ (percentage of RNA fragments above 200 nucleotides) values. The degradation of target reference gene mRNAs (RPL17, GAPDH, HMBS) was evaluated using qPCR and ddPCR. A statistically significant reduction (p < 0.05) of RIN, DV₂₀₀ and RPL17-mRNA levels was observed in "in field" cats within the 51-200 ADDs range, compared to controls. In the "refrigerated" cats, similar degradation patterns were found between 14- and 28-day cats compared to controls (RIN, DV₂₀₀, and qPCR results on GAPDH-mRNA, p < 0.01). RIN and DV₂₀₀ appear to be promising parameters for PMI estimation in cats, particularly for distinguishing recent deaths from those exceeding 50 ADDs.