Multivariate analysis of RNA levels from postmortem human brains as measured by three different methods of RT-PCR. Stanley Neuropathology Consortium.
The analysis of RNA from postmortem human brain tissue by reverse transcription-polymerase chain reaction (RT-PCR) provides a practical method to measure both normal and abnormal brain gene expression. A major limitation in using human material is that yields can vary dramatically from individual to individual, making comparisons between samples difficult. In this report, we study the association of pH and several pre- and postmortem factors on the RNA yields from 89 postmortem human occipital cortices. Glyceraldehyde phosphate dehydrogenase (GAPdH) mRNA levels were measured by RT-PCR. A major variant in this method is the priming used in the reverse transcription reaction. Three different methods of reverse transcription were performed and the resultant levels of products compared against the pre- and postmortem factors and pH. The levels of GAPdH correlated significantly to pH and pH itself to the rapidity of death (RoD) (agonal state) indicating that premortem factors may play the greatest role in determining postmortem RNA levels. The three methods of priming showed different sensitivities, most notably that oligo dT priming alone is vulnerable to long freezer intervals (FI). We conclude that premortem factors are the major affectors of RNA levels variations and that the polyA tail region of the molecule appears to be adversely affected by extended freezer storage.
Johnston
N L
NL
Stanley Foundation Neurovirology Laboratory, Department of Pediatrics, Johns Hopkins University, Baltimore, MD 21287-4933, USA.
Cervenak
J
J
Shore
A D
AD
Torrey
E F
EF
Yolken
R H
RH
Cerevnak
J
J
D001344:Autopsy; D003710:Demography; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015999:Multivariate Analysis; D009778:Occipital Lobe / Q000737:chemistry*; D010446:Peptide Fragments / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012044:Regression Analysis; D014158:Transcription, Genetic
D010446:Peptide Fragments; D012333:RNA, Messenger; C068142:glyceraldehyde 3-phosphate dehydrogenase (304-313); D005987:Glyceraldehyde-3-Phosphate Dehydrogenases