QUANTIFICATION OF MN-SOD MESSENGER-RNAS BY USING A COMPETITIVE REVERSE-TRANSCRIPTION POLYMERASE CHAIN-REACTION

The manganese superoxide dismutase plays an important role in the cell ular response to oxidative stress and appears to be highly regulated b y many factors. The study of this gene's expression is difficult to ac hieve due to multiple rat Mn-SOD transcripts, In this report we descri bed the quantification of the rat Mn-SOD transcripts by competitive re verse transcription-polymerase chain reaction. The competitor RNA was transcribed from a synthetic gene generated by PCR, This gene was comp osed of the T7 polymerase promoter linked to a 102 base-pairs deleted rat Mn-SOD cDNA. Both the target RNA and the competitor RNA were rever se-transcribed and coamplified with the same primers, AU the rat Mn-SO D mRNAs were simultaneously quantified by amplification of a common re gion. The use of a fluorescent primer led to fluorescent PCR products detected and quantified by the use of an automated DNA sequencer which avoides the use of the radioactivity, Small variations in Mn-SOD mRNA concentration (30%) were determined. This method has been applied to study the expression of Mn-SOD mRNA in rat liver after chronic ethanol feeding, Expression of Mn-SOD transcripts was not modified and did no t account for the increased Mn-SOD activity.