RNA integrity in post-mortem samples: influencing parameters and implications on RT-qPCR assays

Area of Science:

• Molecular Biology
• Genomics
• Post-mortem Studies

Background:

• Post-mortem human tissue offers insights into gene expression near death.
• RNA degradation in post-mortem samples is a significant challenge.
• Factors influencing RNA integrity are not fully understood.

Purpose of the Study:

• To analyze how impaired RNA integrity affects quantitative gene expression data reliability.
• To identify ante- and post-mortem factors reducing RNA integrity in human brain, cardiac, and skeletal muscle tissues.
• To assess the impact of tissue type, age, gender, BMI, agony duration, cause of death, and post-mortem interval on RNA integrity.

Main Methods:

• Quantitative gene expression analysis using reverse transcription quantitative polymerase chain reaction (RT-qPCR).
• Generation of RNA degradation profiles for three gene transcripts.
• Assessment of RNA integrity number (RIN) in 136 post-mortem samples.
• Statistical analysis of various ante- and post-mortem parameters on RNA integrity.

Main Results:

• Impaired RNA integrity significantly affects RT-qPCR results, causing shifts in cycle threshold values.
• Brain tissue showed significantly lower RNA integrity compared to skeletal and cardiac muscle.
• Higher Body Mass Index (BMI > 25) was associated with lower RNA integrity in skeletal muscle.
• Data normalization partially mitigated the effects of impaired RNA quality.

Conclusions:

• RNA degradation in post-mortem samples complicates gene expression analysis, especially for subtle differences.
• Large gene expression differences may still be detectable in low-integrity samples.
• Recommendations include generating degradation profiles for all transcripts to understand assay detection limits in post-mortem studies.