Untitled - CNR

Fishery and Sea ResourcesFigure 2: EROD activity levels (pmol min −1 mg prot −1 ± s.d.) in M. barbatus in threedifferent sites and two sampling periods.spectrophotometric method described byHabig et al. [38] modified for microplatereaders. AChE activity was measured onmicroplate by the method of Ellman et al.[39]. Total proteins were measured accordingto Bradford [40] and total proteinsconcentration was expressed in mg·ml −1 .2.4 Total RNA isolation andreverse transcription polymerasechain reaction (RT-PCR)The expression of the vitellogenin genewas qualitatively investigated on 45 and 14male specimens from the summer and wintersurveys, respectively. In particular, inJune 16 from site S1 (size range 13.5-22cm), 21 from site S2 (size range 15-22),and 8 from the site S3 (size range 15.5-20 cm), and in January 8 from the site S1(size range 12-19.5 cm), 3 from site S2(size range 16-19 cm) and 3 from site 3(size range 12.5-16.5 cm) were collected.The small number of individuals analyzedfor the winter survey was caused by theloss of some samples. Six females, oneper survey and site, were analyzed as positivecontrol. Total RNA from liver tissuewas extracted in Trizol reagent, followingthe procedure recommended by themanufacturer (Invitrogen). The total RNAwas digested with RNase free DNases toremove endogenous DNA contamination.Primer pair sequences, their amplicon sizeand annealing temperatures are shown inTable 1. All primer pairs gave a singleband pattern for the expected amplicon sizein all reactions, and no amplification occurredin RT reactions without enzymes.2086