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High stability of mRNAs postmortem and protocols for their assessment by RT-PCR
K Yasojima1, E G McGeer, P L McGeer
1Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3.
Brain Research. Brain Research Protocols
|December 6, 2001
Summary
Brain mRNA is remarkably stable postmortem, even after 15 years of frozen storage. This stability allows for reliable gene expression analysis in postmortem brain tissue, crucial for neurological research.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Gene expression measurement is vital in brain research.
- Understanding mRNA stability in postmortem tissue is crucial for accurate analysis.
Purpose of the Study:
- To assess the postmortem stability of various brain mRNAs.
- To evaluate the impact of frozen storage and thawing on mRNA integrity.
Main Methods:
- RNA extraction from fresh and frozen brain tissue.
- Complementary DNA (cDNA) synthesis and polymerase chain reaction (PCR) amplification.
- Quantification of specific messenger RNAs (mRNAs) including cyclophilin, C3, C4, MAP-2, and COX-2.
Main Results:
- Little RNA deterioration observed in postmortem tissue up to 96 hours.
- No degradation from 15 years of frozen storage (-70°C), but gradual deterioration post-thawing.
- mRNA stability varied post-thawing: Cyclophilin, C3, C4 stable for 8 hours; MAP-2 and COX-2 degraded significantly within 2-4 hours, with COX-2 showing drastic loss by 8 hours.
Conclusions:
- Postmortem brain RNA exhibits significant stability, even with extended autolysis times if kept cold.
- Frozen brain tissue maintains mRNA integrity for 15 years, with a limited window of stability (1-2 hours) after thawing.
- Reliable gene expression studies are feasible using postmortem brain tissue under controlled conditions.