Quantitative PCR assays to measure the HPI axis neuropeptides corticotropin-releasing factor (CRF) and urotensin I (UI) in channel catfish (Ictalurus punctatus)

Abstract

Central to the stress response of fishes is the upregulation of the hypothalamus-pituitary-interrenal (HPI) axis. This pathway is initiated by an increase of corticotropin-releasing factor (CRF) and urotensin I (UI) transcripts which translate into hormones stimulating the production of cortisol in the anterior kidney. Quantifying changes in CRF and UI transcription may provide molecular-level evidence of the initiation of the stress response. To determine changes in CRF and UI transcription in the channel catfish (Ictalurus punctatus) hypothalamus, two duplex quantitative PCR (qPCR) assays were developed, optimized, and tested for both CRF and UI as targets. Performance of each respective qPCR was validated as a singleplex reaction and a duplex using α-tubulin as a reference. There were no significant declines in qPCR efficiency observed in the duplex format for either assay. These duplex qPCR assays are advantageous over single-gene reactions by reducing the number of wells used on the PCR plate, reducing reagent consumption, and minimizing sources of experimental error. Further, these assays have potential to serve as valuable tools evaluating the stress response of channel catfish at the molecular level.