Fig. 3. Estimate of Na⁺+K⁺-ATPase -subunit mRNA abundance in total RNA extracts of anterior and posterior gills of Callinectes sapidus acclimated for at least 2 weeks to 35 or 5  salinity. Gills 3–5 (anterior) and 6–8 (posterior) were pooled from three individuals for each RNA preparation. mRNA levels were evaluated by duplex quantitative RT-PCR under conditions of limiting template, with arginine kinase mRNA serving as an invariant standard (Kotlyar et al., 2000). The primers employed to amplify Na⁺+K⁺-ATPase -subunit cDNA were NAK10F and NAK16R and for arginine kinase AKF5 (5'-CGCTGAGTCTAAGAAGGGATT-3') and AKCALLR1 (5'-CCCAGGCTTGTCTTCTTGTCC-3'). Biotinylated PCR products were visualized after 22 cycles using a streptavidin/alkaline phosphatase procedure (Phototope, New England Biolabs). The data are representative of three separate experiments.