Recovery of high-integrity mRNA from brains of rats killed by high-energy focused microwave irradiation.
Following decapitation, during the brief time period of postmortem brain tissue collection, significant changes in neuro-metabolite levels can occur. To circumvent such changes, we routinely kill rats using 1 to 2 sec pulses of focused high-energy microwave irradiation (10 kW). The effects of high-energy microwave irradiation on total RNA and mRNA integrity in brain however, are unknown. Total RNA recovery, per gram wet weight, in brain regions of microwaved rats was less than 50% of that in rats killed by decapitation. Formaldehyde agarose gel electrophoresis showed that ribosomal RNA components were highly degraded in all brain regions of microwaved rats. In contrast, poly A+mRNA, as measured by poly A+driven cDNA synthesis and Northern analysis, in brain samples of microwaved or decapitated rats was of equal integrity and quantity when expressed per mg tissue weight. Furthermore, positive RT-PCR products for GAPDH and TNF-alpha were observed in brain regions of both microwaved and decapitated rats. These observations indicated that high-energy focused microwave irradiation does not reduce mRNA abundance and integrity. Thus, this method of animal sacrifice can be used to simultaneously study, accurately and precisely, levels of brain metabolites as well as molecular biological events in discrete brain regions of experimental animals without postmortem interference.
Mayne
M
M
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, 753 McDermot Avenue, Winnipeg, Canada.
Shepel
P N
PN
Geiger
J D
JD
D000241:Adenosine / Q000302:isolation & purification; D000818:Animals; D015152:Blotting, Northern; D001923:Brain Chemistry / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D008297:Male; D008872:Microwaves; D011180:Postmortem Changes; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics
D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000241:Adenosine