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 hemocyt es. The assay was based on titration of a competitive external standard cRN A 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 usi ng biotinylated primers common to both target and standard sequences. Ampli fied products were bound to streptavidin-coated plates and hybridized to se quence-specific fluorescein-labeled probes. Detection was based on single p hoton counting of chemiluminescence generated by an alkaline phosphatase-co njugated antifluorescein antibody. For quantification, the target chemilumi ncescence 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 mR NA equivalent to that in 180 hemocytes was easily detected, and, for routin e quantitative analysis, was sufficiently sensitive to quantify basal and i nduced MT mRNA. Basal hemocyte MT mRNA of 133 +/- 8 (1 S.E.) amol per micro gram total RNA was induced 5-fold to 573 +/- 14 amol per microgram total RN A by in vitro exposure to 15 muM CdCl2 for 20 h. (C) 2000 Elsevier Science B.V. All rights reserved.