The forgotten variable? Does the euthanasia method and sample storage condition influence an organisms transcriptome
B Chakkingal Bhaskaran1, R Meyermans2, W Gorssen2
1Department of Biosystems, Centre for Animal Breeding and Genetics, KU Leuven, Kasteelpark Arenberg 30, Box 2472, Leuven, 3001, Belgium. bimalcb98@gmail.com.
BMC Genomics
|December 13, 2023
Summary
Nitrogen gas euthanasia (ANOXIA) is a suitable alternative for piglets in transcriptomic research. However, both euthanasia methods and tissue storage conditions can impact gene expression, requiring careful consideration to avoid misinterpreting results.
Area of Science:
- Animal research methodology
- Molecular biology
- Transcriptomics
Background:
- Transcriptomic studies necessitate fresh tissue collection post-euthanasia.
- Euthanasia methods can introduce unintended gene expression variations.
- Investigating the impact of euthanasia and storage on gene expression is crucial for reliable transcriptomic data.
Purpose of the Study:
- To compare nitrogen gas (ANOXIA) versus T-61® (T61) euthanasia methods for piglet transcriptomic research.
- To analyze the effects of RNAlater™ (RL) and liquid nitrogen (LN2) snap freezing on gene expression profiles.
- To identify potential sources of biological variation in transcriptomic studies.
Main Methods:
- 3'mRNA-Seq data analysis of pituitary, hypothalamus, liver, and lung tissues.
- Comparison of gene expression profiles between different euthanasia methods.
- Assessment of RNA integrity and gene expression under two common tissue storage conditions (RL and LN2).
Main Results:
- No significant differential expression in protein-coding genes was observed between ANOXIA and T61 euthanasia methods.
- Elevated levels of small nuclear RNAs (snRNAs) were detected in pituitary samples from the ANOXIA group.
- RNAlater™ (RL) provided superior RNA preservation (higher RIN values) but influenced gene expression differently than liquid nitrogen (LN2) snap freezing.
Conclusions:
- Nitrogen anoxia is a viable euthanasia method for piglet transcriptomic studies, with minimal impact on protein-coding genes.
- Non-protein coding RNAs, such as snRNAs, may be affected by euthanasia method, potentially influencing downstream gene expression.
- Both euthanasia and tissue storage methods represent significant sources of biological variation that must be accounted for to prevent misinterpretation of transcriptomic data.
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