Linking mechanistic and behavioral responses to sublethal esfenvalerate exposure in the endangered delta smelt; Hypomesus transpacificus (Fam. Osmeridae)

Connon, Richard E; Geist, Juergen; Pfeiff, Janice; Loguinov, Alexander V; D'Abronzo, Leandro S; Wintz, Henri; Vulpe, Christopher D; Werner, Inge

A total of 1.5 μg RNA was cDNA synthesized using random primers, and diluted to a total of 50 μl with nuclease free water to generate sufficient template for qPCR analysis. TaqMan Universal PCR Mastermix (Applied Biosystems) was used in q-PCR amplifications in a reaction containing 10mMTris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 2.5 mM deoxynucleotide triphosphates, 0.625 U AmpliTaq Gold DNA polymerase per reaction, 0.25 U AmpErase UNG per reaction and 5 μL of cDNA sample in a final volume of 12 μL. The samples were placed in 384 well plates and cDNA was amplified in an automated fluorometer (ABI PRISM 7900 Sequence Detection System, Applied Biosystems). Amplification conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C and 60 s at 60°C. Fluorescence of samples was measured every 7 s and signals were considered positive if fluorescence intensity exceeded 10 times the standard deviation of the baseline fluorescence (threshold cycle, CT). SDS 2.2.1 software (Applied Biosystems) was used to quantify transcription.