Midori green advance

Male offspring were sacrificed by decapitation on PND 7, and brain regions, including the hippocampus, cerebral cortex, cerebellum, and olfactory bulb, were quickly separated, and stored at -80°C until analysis by RT-PCR. The total RNA was isolated from the each brain region using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). The cDNA for a given mRNA was synthesized using oligo-dT and random hexamers with a Primescript RT reagent kit (Takara Bio). Expression of AhR and GAPDH mRNA were determined using Veriti thermal cycler (Applied Biosystems) with KOD Plus kit (Toyobo, Osaka, Japan). The amplification conditions were as follows: 95°C for 1 min; and 30 cycles of 95°C for 15 s, 55°C for 15 s, and 68°C for 15 s. The PCR primers for amplifying the murine AhR and GAPDH mRNA were 5′-tgtagagcacaaatcagaga-3′/5′-gatagtggaggaagcatag-3′, and 5′-acccagaagactgtggatgg-3′/5′-cacattgggggtaggaacac-3′, respectively. The 25-μl reaction solution contained 250 nM each primer, 1 × KOD Plus buffer, 200 μM dNTP mixture, 1 mM MgSO₄, and 0.5 U of KOD Plus DNA polymerase. PCR products were separated by electrophoresis on agarose gels that were stained with Midori Green Advance (Nippon Gene, Tokyo, Japan). PCR products of AhR and GAPDH mRNA were expected to have 123 and 171 bp in size, respectively.

Developing mice at P3 and P5 were decapitated, and several organs, including the brain, liver, lung, kidney, thymus, and spleen, were quickly removed and stored at −80 °C until RT-PCR analysis. The total RNA was isolated from each organ using an RNeasy Mini Kit (Qiagen, Tokyo, Japan). The cDNA for a given mRNA was synthesized using oligo-dT and random hexamers with a Primescript RT reagent kit (Takara Bio). Expression levels of Ahr and Gapdh transcripts were determined using a Veriti thermal cycler (Applied Biosystems) with a KOD Plus kit (Toyobo, Osaka, Japan). The amplification conditions were as follows: 95 °C for 1 min, followed by 35 cycles of 95 °C for 15 s, 55 °C for 15 s, and 68 °C for 30 s. The PCR primers for amplifying the murine Ahr and Gapdh transcripts were 5′-AGGATTTGCAAGAAGGAGAG-3′/5´-TTGGTTCGAATTTCCAGGAT-3´ and 5′-ACCCAGAAGACTGTGGATGG-3′/5′-CACATTGGGGGTAGGAACAC-3′, respectively. The 20-μl reaction solution contained 400 nM of each primer, 1× KOD Plus buffer, 200 μM dNTP mixture, 1 mM MgSO₄, and 0.5 U of KOD Plus DNA polymerase. PCR products were separated by electrophoresis on agarose gels, which were stained with Midori Green Advance (Nippon Gene). The PCR products of the Ahr and Gapdh transcripts were expected to be 508 and 171 bp in size, respectively.