QUANTITATION OF METALLOTHIONEIN MESSENGER-RNA BY RT-PCR AND CHEMILUMINESCENCE

A general procedure has been developed for the determination of mRNA e xpression by reverse transcription polymerase chain reaction (RT-PCR), over a wide concentration range, with quick quantitation of amplified products by luminescence. The discriminating power of this approach i s the specific hybridization of PCR product to ruthenium-labeled oligo nucleotide probe(s). This method is sensitive enough to detect increas es in the formation of PCR product by the 6th cycle. The accumulation of PCR product was successfully modeled with a recursive relationship. This procedure was capable of accurately determining starting templat e copies over a 9-log dynamic range, with a sensitivity limit of 10(2) copies. Inclusion of an mRNA internal standard (identical to amplifie d template except for a 6-bp deletion) corrected variabilities in the reverse transcriptase as well as PCR, allowing for the expression of d ata as mRNA copy number/mu g total tissue RNA. This procedure was used to detect changes in levels of winter flounder (Pleuronectes american us) liver metallothionein mRNA. Liver metallothionein mRNA levels rang ed from 1.0 x 10(6) copies/mu g total tissue RNA in control samples to 1.0 x 10(9) copies/mu g total tissue RNA in samples treated with Cd ( a known metallothionein mRNA inducer).