A Comparative Study on the Effect of Euthanasia Methods and Sample Storage Conditions on RNA Yield and Quality in Porcine Tissues

Animals have been used as subjects in biomedical research for a long time. In these studies, tissue collection for RNA profiling is often essential, and hence, the animals are euthanised using standard injectable anaesthetics such as sodium pentobarbital or T-61((R)). Recently, a euthanasia method using an inhalant anaesthetic 'nitrogen gas in foam' (ANOXIA (TM)) has gained further interest as it is claimed to be more animal-friendly. However, little is known on its effect on subsequent RNA analysis. There are also no studies on the interaction effect of these euthanasia methods and different tissue storage conditions such as RNAlater (TM) or snap freezing using liquid nitrogen on RNA measurements. It is very important to investigate if the choice of euthanasia method in research animals as well as the tissue storage condition impact RNA measurements. The current study compared the two euthanasia methods in male piglets and found that the nitrogen anoxia technique (ANOXIA (TM)) could be a suitable alternative to T-61((R)) based on RNA quality parameters because no differences were detected. Storage in RNAlater (TM) significantly increased the RNA integrity in comparison to snap freezing, but no interaction effect of the euthanasia methods and storage conditions on RNA measurements were detected. Animals used in research often have to be euthanised, especially when tissue sampling is essential. Recently, a euthanasia method, utilizing an inhalant anaesthetic 'nitrogen gas in foam' in an anoxia box (ANOXIA (TM)), has gained considerable interest as it claimed to be more animal-friendly. However, it is not clear whether the use of this euthanasia method has an influence on RNA measurements. Moreover, there are no studies on the interaction effect of different euthanasia methods on the tissue sample storage conditions. The current study compared RNA measurements from two euthanasia methods (ANOXIA (TM) vs. T-61((R)) injection) and two storage conditions (RNAlater (TM) vs. snap freezing) in 12 male piglets. The nitrogen anoxia method had a significantly higher RNA yield (p < 0.01) compared to the T-61((R)) method. However, no effect of the euthanasia methods on the A260/230 ratio and RIN value was observed. Tissues stored in RNAlater (TM) had significantly higher RIN values (p < 0.001) compared to snap frozen samples. The present study could not find a significant difference between the two euthanasia methods in piglets, with regard to RNA quality measurements. Hence, the nitrogen anoxia technique (ANOXIA (TM)) might be considered as a suitable alternative to T-61((R)) for euthanasia of piglets used in research.

Automated summary

This study compared the effects of two euthanasia methods (nitrogen gas and injection) on RNA measurements and the interaction effects with two different tissue storage conditions on RNA measurements. The findings suggest that the nitrogen gas method is a suitable alternative to injection for euthanasia, and RNAlater storage condition can significantly improve RNA integrity.