Quantitative reverse transcription polymerase chain reaction of a molluscan metallothionein mRNA

A quantitative assay based on competitive reverse transcription polymerase chain reaction (RT-PCR) was developed for metallothionein (MT) mRNA of the mollusc Crassostrea virginica and applied to analysis of MT mRNA of hemocytes. The assay was based on titration of a competitive external standard cRNA derived from the coding region of the oyster MT mRNA. Serial dilutions of the cRNA standard were coamplified with a constant amount of total RNA using biotinylated primers common to both target and standard sequences. Amplified products were bound to streptavidin-coated plates and hybridized to sequence-specific fluorescein-labeled probes. Detection was based on single photon counting of chemiluminescence generated by an alkaline phosphatase-conjugated antifluorescein antibody. For quantification, the target chemiluminescence was normalized to that of the standard, and the amount of target MT mRNA in the sample was derived from the titration. Cadmium-induced MT mRNA equivalent to that in 180 hemocytes was easily detected, and, for routine quantitative analysis, was sufficiently sensitive to quantify basal and induced MT mRNA. Basal hemocyte MT mRNA of 133+/-8 (1 S.E.) amol per microgram total RNA was induced 5-fold to 573+/-14 amol per microgram total RNA by in vitro exposure to 15 microM CdCl(2) for 20 h.