High stability of mRNAs postmortem and protocols for their assessment by RT-PCR.
Measurement of gene expression is a major area of brain research. We report on the remarkable postmortem stability of a selection of brain mRNAs in both fresh and frozen brain tissue. We describe techniques for extracting total RNA, synthesizing cDNAs from the mRNAs, amplifying specific cDNAs by the polymerase chain reaction technique, and quantitating the products. We chose five genes to study: the housekeeping gene cyclophilin; the complement components C3 and C4; the microtubule associated protein-2 (MAP-2); and the strongly inducible cyclooxygenase COX-2. We found little deterioration in total RNA or in any of the mRNAs in postmortem tissue up to 96 h. When tissue was frozen, stored at -70 degrees C for 15 years and then thawed, there was no evidence of deterioration from storage, but there was gradual deterioration post thawing. All the mRNAs were stable for 1-2 h at 4 degrees C following thawing. Cyclophilin, C3 and C4 mRNAs were still stable after 8 h, MAP-2 and COX-2 mRNAs showed significant deterioration between 2 and 4 h, and COX-2 mRNA showed drastic deterioration between 4 and 8 h. The data give no indication of rapid postmortem degeneration of RNA. Reliable mRNA values may be obtained from postmortem brain with long autolysis times provided the tissue has been kept in the cold, and from frozen tissues for 1-2 h after thawing.
Yasojima
K
K
Kinsmen Laboratory of Neurological Research, Department of Psychiatry, University of British Columbia, 2255 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3.
McGeer
E G
EG
McGeer
P L
PL
D000544:Alzheimer Disease / Q000378:metabolism; D001923:Brain Chemistry / Q000502:physiology*; D003176:Complement C3 / Q000096:biosynthesis; D003181:Complement C4 / Q000096:biosynthesis; D051546:Cyclooxygenase 2; D021983:Cyclophilins / Q000096:biosynthesis; D015180:Electrophoresis, Gel, Two-Dimensional; D004720:Endonucleases / Q000737:chemistry; D004996:Ethidium; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000096:biosynthesis; D008565:Membrane Proteins; D011180:Postmortem Changes; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D012313:RNA; D012333:RNA, Messenger / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D003176:Complement C3; D003181:Complement C4; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007527:Isoenzymes; D008565:Membrane Proteins; C034477:RNA primers; D012333:RNA, Messenger; D012313:RNA; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D004720:Endonucleases; D021983:Cyclophilins; D004996:Ethidium