Real-Time PCR Method for the Quantification of the Two Isoforms of Metallothionein in Lake Trout (Salvelinus namaycush)

Metallothioneins (MTs) are low-molecular-weight proteins whose physiologic roles are the regulation of essential metals Cu and Zn, sequestration of heavy metals, and free radical scavenging. Induced production of MTs in a wide variety of organisms exposed to heavy metals has made them popular exposure indicators. While it has been postulated that the three different isoforms of MT play different physiologic roles, methods to discern induction separately have not been available. The development of real-time polymerase chain reaction (real-time PCR) primers and TaqMan probes to measure the two MT isoforms found in salmonid fish are described. Assuming a high degree of homology between the isoforms and within different groups of salmonids, the sequences for MT-I and MT-II from rainbow trout were used to develop primers and probes for lake trout using the Primer3 program. Two sections of each isoform that varied by only a few nucleotides were targeted. SYBR Green validated the primer specificity, and melt curve analysis further ensured that only one product was amplified. Analysis of archived samples from fish captured in unmanipulated reference lakes or from lakes experimentally treated with cadmium or ethynylestradiol (EE2) afforded an examination of seasonal and contaminant influences on MT-I and MT-II mRNA expression.