Quantification of Mn-SOD mRNAs by using a competitive reverse-transcription polymerase chain reaction

SUMMARY

The manganese superoxide dismutase plays an important role in the cellular response to oxidative stress and appears to be highly regulated by many factors. The study of this gene's expression is difficult to achieve due to multiple rat Mn-SOD transcripts. In this report we described the quantification of the rat Mn-SOD transcripts by competitive reverse transcription-polymerase chain reaction. The competitor RNA was transcribed from a synthetic gene generated by PCR. This gene was composed 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 reverse-transcribed and coamplified with the same primers. All the rat Mn-SOD mRNAs were simultaneously quantified by amplification of a common region. 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 not account for the increased Mn-SOD activity.