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Published on: October 21, 2011
Prolonged Sample Storage Reshapes the m6A Methylation Landscape Through RNA Degradation
Lingsong Yao1, Zhiyu Liu1, Ying Wang1
1State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 210096, China.
Abstract:
N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA, essential for post-transcriptional regulation. MeRIP-seq is widely used for m6A profiling, but RNA degradation challenges accurate analysis. While the effects of sample handling on RNA are known, the impact of tissue sample storage durations on m6A remains unclear. We investigated how sample storage durations (0, 2, 12, 24, and 48 h at room temperature) affect RNA integrity, m6A peaks, and transcriptomes in mouse liver. RNA integrity declined with time, reducing m6A peak number and reproducibility. Prolonged storage diverged m6A profiles, increased unreported peaks, shifted RRACH motifs, and redistributed peaks to intergenic/intronic regions. Integrated data showed opposing changes in m6A and expression for some genes, suggesting storage degradation confounds epitranscriptomic interpretation. Sample storage durations are critical for m6A accuracy, emphasizing the need for standardized handling in MeRIP-seq studies.
Insights
Sample storage duration significantly impacts N6-methyladenosine (m6A) profiling accuracy. Prolonged storage degrades RNA, altering m6A peaks and transcriptomes, necessitating standardized handling for reliable epitranscriptomic studies.
Area of Science:
- Epitranscriptomics
- Molecular Biology
- RNA Modifications
Background:
- N6-methyladenosine (m6A) is a crucial mRNA modification regulating gene expression.
- MeRIP-seq is a common method for m6A profiling, but RNA degradation poses analytical challenges.
- The effect of tissue sample storage duration on m6A profiling accuracy is not well understood.
Purpose of the Study:
- To investigate the impact of varying room temperature storage durations (0-48 hours) on mouse liver RNA integrity and m6A profiles.
- To assess how storage time affects MeRIP-seq data quality, peak detection, and transcriptome analysis.
- To determine the critical storage time limits for accurate m6A epitranscriptomic profiling.
Main Methods:
- Mouse liver tissue samples were stored at room temperature for 0, 2, 12, 24, and 48 hours.
- RNA integrity was assessed using standard methods.
- m6A profiling was performed using MeRIP-seq, followed by bioinformatic analysis of peak distribution, motif enrichment, and correlation with gene expression.
Main Results:
- RNA integrity significantly decreased with increasing storage duration.
- m6A peak number and reproducibility declined over time.
- Prolonged storage led to altered m6A profiles, increased spurious peaks, shifted RRACH motifs, and redistribution of peaks to non-coding regions.
- Storage-induced degradation introduced confounding effects, showing opposing trends between m6A levels and gene expression for certain genes.
Conclusions:
- Tissue sample storage duration is a critical factor influencing the accuracy and reliability of m6A profiling by MeRIP-seq.
- Degradation artifacts from prolonged storage can lead to misinterpretation of epitranscriptomic data.
- Standardized sample handling protocols are essential for robust m6A research.
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