Prolonged Sample Storage Reshapes the m⁶A Methylation Landscape Through RNA Degradation.

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.