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Fig. 1. Metallothionein-2A (MT-2A) gene expression by CEM-C12 cells exposed to water samples. Cells were incubated (1.5 x 106 cells mL–1) for (A) 6 and (B) 18 h in medium reconstituted with either water samples (1–10) or ultrapure water as control (C) as described in the Materials and Methods section. Cadmium chloride (5 µM) was used as a positive control (Cd). Total cellular RNA was extracted and subjected to reverse transcriptase–polymerase chain reaction (RT–PCR) analysis. As described in the Material and Methods section, [
-32P]-dCTP was added to the PCR mixture and quantification of PCR products was done by measuring the amount of 32P-radiolabeled nucleotide (cpm) incorporated into amplified fragments. The 32P-labeled bands of PCR products corresponding to MT-2A and glyceraldehyde-3'-phosphate dehydrogenase (GaPDH; housekeeping gene) are presented. (C) For 18 h of exposure, MT-2A gene expression was evaluated by the signal ratio of cpm values for MT-2A on respective cpm values for GaPDH. The RT–PCR analysis was performed in triplicate (standard deviation was always less than 15% of the mean). Results are expressed as fold of induction over control (signal ratio from cells cultured with water sample to signal ratio from cells cultured with ultrapure water).
