Dynamic changes in RNA integrity, gene expression, and tissue pathomorphology of experimental mice in the postmortem period

Aim. To examine the pattern of morphological changes, RNA quality number, and gene expression in mouse tissues sampled at autopsy under controlled experimental conditions.

Materials and methods. Balb/c mice were euthanized and subsequently subjected to necropsy at 0, 3, 12, 24, 48, and 72 hours of the postmortem period. During the first three hours following euthanasia, the mice were maintained at room temperature, after which they were transferred to a refrigerator (4 º С). Total RNA was extracted from tissue samples taken from the kidney, liver, and brain; the integrity of the RNA samples was assessed by capillary electrophoresis, and the RNA quality number (RQN) was calculated. The expression levels of Actb, Epas1, and Rps18 housekeeping genes were evaluated by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) with original primers and probes using the TaqMan assay. The histologic examination was performed according to standard techniques.

Results. Degradation of RNA extracted from mouse kidney tissues appeared to be greater than that of RNA taken from the liver. In the meantime, a negative linear correlation was observed between RQN and the duration of the postmortem interval for liver and kidney samples. In contrast, no significant changes in the RQN score were observed for brain RNA samples at any of the time points. The expression of the Epas1 and Rps18 genes was significantly decreased in mouse kidney and liver tissues. However, the level of Epas1 and Rps18 gene expression in the brain remained stable at all time points and did not exhibit a significant decrease at 72 hours after euthanasia. No obvious morphological changes were detected by the histologic examination, which does not exclude the presence of ultrastructural pathological changes.

Conclusion. RQN in autopsy tissues serves as a crucial predictor of sample quality for molecular biology studies, including gene expression analysis.

1. Mackenzie I.R., Neumann M. Molecular neuropathology of frontotemporal dementia: insights into disease mechanisms from postmortem studies. Journal of Neurochemistry. 2016;38:54–70. DOI: 10.1111/jnc.13588.

2. Zhu Y., Wang L., Yin Y., Yang E. Systematic analysis of gene expression patterns associated with postmortem interval in human tissues. Scientific Reports. 2017;7(1):5435. DOI: 10.1038/s41598-017-05882-0.

3. Strand C., Enell J., Hedenfalk I., Fernö M. RNA quality in frozen breast cancer samples and the influence on gene expression analysis – a comparison of three evaluation methods using microcapillary electrophoresis traces. BMC Molecular Biology. 2007;8:1–9. DOI: 10.1186/1471-2199-8-38.

4. Kocsmár É., Schmid M., Cosenza-Contreras M., Kocsmár I., Föll M., Krey L. et al. Proteome alterations in human autopsy tissues in relation to time after death. Cellular and Molecular Life Sciences. 2023;80(5):117. DOI: 10.1007/s00018-023-04754-3.

5. Cao L., Huang C., Zhou D.C., Hu Y., Lih T.M., Savage S.R. et al. Proteogenomic characterization of pancreatic ductal adenocarcinoma. Cell. 2021;184(19):5031–5052. DOI: 10.1016/j.cell.2021.08.023.

6. Sidova M., Tomankova S., Abaffy P., Kubista M., Sindelka R. Effects of post-mortem and physical degradation on RNA integrity and quality. Biomolecular Detection and Quantification. 2015;5:3–9. DOI: 10.1016/j.bdq.2015.08.002.

7. Johnson E.S., Stenzel K.E., Lee S., Blalock E.M. Declining RNA integrity in control autopsy brain tissue is robustly and asymmetrically associated with selective neuronal mRNA signal loss. BioRxiv. 2021;2021. DOI: 10.1101/2021.09.07.459326.

8. Fan J., Khani R., Sakamot H., Zhon Y., Michae C., Pen D. et al. Quantification of nucleic acid quality in postmortem tissues from a cancer research autopsy program. Oncotarget. 2016;7(41):66906. DOI: 10.18632/oncotarget.11836.

9. White K., Yang P., Li L., Farshori A., Medina A.E., Zielke H.R. Effect of postmortem interval and years in storage on RNA quality of tissue at a repository of the NIH NeuroBioBank. Biopreservation and Biobanking. 2018;16(2):148–157. DOI: 10.1089/bio.2017.0099.

10. Van der Linden A., Blokker B.M., Kap M., Weustink A.C., Riegman P.H., Oosterhuis J.W. Post-mortem tissue biopsies obtained at minimally invasive autopsy: an RNA-quality analysis. PLoS One. 2014;9(12):e115675. DOI: 10.1371/journal.pone.0115675.

11. Miyahara K., Hino M., Yu Z., Ono C., Nagaoka A., Hatano M. et al. The influence of tissue pH and RNA integrity number on gene expression of human postmortem brain. Frontiers in Psychiatry. 2023;14:1156524. DOI: 10.3389/fpsyt.2023.1156524.

12. Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research. 2001;29(9):e45–45. DOI: 10.1093/nar/29.9.e45/

13. Schroeder A., Mueller O., Stocker S., Salowsky R., Leiber M., Gassmann M. et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology. 2006;7:1–14. DOI: 10.1186/1471-2199-7-3.

14. Thompson K.L., Pine P.S., Rosenzweig B.A., Turpaz Y., Retief J. Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA. BMC Biotechnology. 2007:7:1–12. DOI: 10.1186/1472-6750-7-57.

15. Weickert C.S., Sheedy D., Rothmond D.A., Dedova I., Fung S., Garrick T. et al. Selection of reference gene expression in a schizophrenia brain cohort. Australian & New Zealand Journal of Psychiatry. 2010;44(1):59–70. DOI: 10.3109/00048670903393662.

16. Hostiuc S., Rusu M.C., Mănoiu V.S., Vrapciu A.D., Negoi I., Popescu M.V. Usefulness of ultrastructure studies for the estimation of the postmortem interval. A systematic review. Rom. J. Morphol. Embryol. 2017;58(2):377–384.

17. Kvastad L., Carlberg K., Larsson L., Villacampa E.G., Stuckey A., Stenbeck L. et al. The spatial RNA integrity number assay for in situ evaluation of transcriptome quality. Communications Biology. 2021;4(1):57. DOI: 10.1038/s42003-020-01573-1.

18. Vennemann M., Koppelkamm A. mRNA profiling in forensic genetics I: possibilities and limitations. Forensic Science International. 2010;203(1-3):71–75. DOI: 10.1016/j.forsciint.2010.07.006.

19. Stan A.D., Ghose S., Gao X.M., Roberts R.C., Lewis-Amezcua K., Hatanpaa K.J. et al. Human postmortem tissue: what quality markers matter? Brain Research. 2006;1123(1):1–11. DOI: 10.1016/j.brainres.2006.09.025.

20. Sobue S., Sakata K., Sekijima Y., Qiao S., Murate T., Ichihara M. Characterization of gene expression profiling of mouse tissues obtained during the postmortem interval. Experimental and Molecular Pathology. 2016;100(3):482–492. DOI: 10.1016/j.yexmp.2016.05.007.

21. Heimberger A.B., Crotty L.E., Archer G.E., McLendon R.E., Friedman A., Dranoff G. et al. Bone marrow-derived dendritic cells pulsed with tumor homogenate induce immunity against syngeneic intracerebral glioma. Journal of Neuroimmunology. 2020;103(1):16–25. DOI: 10.1016/s0165-5728(99)00172-1.

22. Padhi B.K., Singh M., Rosales M., Pelletier G., Cakmak S. A PCR-based quantitative assay for the evaluation of mRNA integrity in rat samples. Biomolecular Detection and Quantification. 2018;15:18–23. DOI: 10.1016/j.bdq.2018.02.001.

23. Fleige S., Pfaffl M.W. RNA integrity and the effect on the real-time qRT-PCR performance. Molecular Aspects of Medicine. 2006;27(2-3):126–139. DOI: 10.1016/j.mam.2005. 12.003.