High stability of mRNAs postmortem and protocols for their assessment by RT-PCR.

Author(s): Yasojima K, McGeer EG, McGeer PL

Publication: Brain Res Brain Res Protoc, 2001, Vol. 8, Page 212-8

PubMed ID: 11733198 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if prolonged frozen storage of a brain specimen (15 years at -70 degrees C) or post thaw storage at 4 degrees C (0-24 h) affects RNA quality or mRNA expression levels of 5 representative genes: cyclophilin, C3, C4, MAP-2, COX-2.

   Summary of Findings:

   As determined by OD 260/280 ratios, the quality of total RNA was not affected by prolonged frozen storage (15 years at -70 degrees C), compared to freshly obtained autopsy specimens, when processed immediately after thawing. Total RNA extracted from specimens stored for 15 years at -70 degrees C showed significant degradation after 4 h of post-thaw specimen storage at 4 degrees C and extensive degradation after 24 h. Despite evidence of degradation, cyclophilin, C3 and C4 were not markedly affected by post thaw storage at 4 degrees C. COX-2 expression exhibited a robust decrease in expression after 8 h of post thaw specimen storage at 4 degrees C, while MAP-2 expression exhibited a slight decrease in expression after 4 h of storage.

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Frozen

   Diagnoses:

   • Alzheimer's Disease
   • Autopsy

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Postmortem interval	7 h
   RT-PCR Specific	Targeted nucleic acid	Cyclophilin C3 C4 MAP-2 COX-2
   Storage	Post-thaw duration	0 h 1 h 2 h 4 h 8 h 24 h
   Storage	Storage temperature	-70 degrees C (15 yr) 4 degrees C

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2. Study Purpose

   The purpose of this study was to determine if prolonged specimen storage at 4 degrees C prior to freezing affects RNA quality.

   Summary of Findings:

   Specimens stored at 4 degrees C for up to 144 h prior to freezing did not exhibit significant RNA degradation. Total RNA, extracted from a specimen stored at 4 degrees C for 144 h, was stored at -80 degrees C for 3 years, and showed gene expression levels comparable to promptly frozen brain specimens extracted immediately after prolonged storage.

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Frozen

   Diagnoses:

   • Alzheimer's Disease
   • Autopsy
   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Spectrophotometry
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   RT-PCR Specific	Targeted nucleic acid	Cyclophilin C3 C4 MAP-2 COX-2
   Storage	Storage duration	24 h 48 h 144 h 3 yr
   Storage	Storage temperature	4 degrees C -80 degrees C
   Preaquisition	Postmortem interval	7 h

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