To examine the pattern of ac-AKH expression, tissues were collected from control animals previously injected with 500 μL ASW (from Experiment 1). The central nervous system (CNS), heart, kidney, gill, esophagus, crop, and intestine were harvested, snap-frozen on dry ice, and stored at −70°C until RNA isolation. Total RNA was isolated using TRIzol reagent (Ambion, Austin, TX) according to the manufacturer's instructions. cDNA synthesis was performed from 1 μg total RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Germantown, MD) according to the manufacturer's instructions.
For quantitative reverse-transcription PCR (qPCR), A. californica actin (ac-actin) was used as a reference gene to normalize the target gene expression. qPCR primers for ac-akh were designed based on the previously published sequence (11 (link)). qPCR of ac-akh was carried out using FastStart Universal SYBR Green mix (Roche, Indianapolis, IN) and calculated using the 2−ΔΔCT method (26 (link)). Each reaction contained 9 μL SYBR Green mix, 9 μL ultrapure water, 0.5 μM gene-specific primers, and 2 μL (for ac-actin and ac-akh) of cDNA. Forward and reverse primers were 5′-CACACTGTCCCCATCTACGA and 5′-CCAGCGAGATCCAATCTCAT for ac-actin, and 5′-TTAATACAGCGAACCGCAAA, and 5′-TCACAGTTCTGGGCAGGTATT for ac-akh.
For quantitative reverse-transcription PCR (qPCR), A. californica actin (ac-actin) was used as a reference gene to normalize the target gene expression. qPCR primers for ac-akh were designed based on the previously published sequence (11 (link)). qPCR of ac-akh was carried out using FastStart Universal SYBR Green mix (Roche, Indianapolis, IN) and calculated using the 2−ΔΔCT method (26 (link)). Each reaction contained 9 μL SYBR Green mix, 9 μL ultrapure water, 0.5 μM gene-specific primers, and 2 μL (for ac-actin and ac-akh) of cDNA. Forward and reverse primers were 5′-CACACTGTCCCCATCTACGA and 5′-CCAGCGAGATCCAATCTCAT for ac-actin, and 5′-TTAATACAGCGAACCGCAAA, and 5′-TCACAGTTCTGGGCAGGTATT for ac-akh.
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