Development of multitissue microfluidic dynamic array for assessing changes in gene expression associated with channel catfish appetite, growth, metabolism, and intestinal health

Authors: SCHROETER, JULIE - Southern Illinois University; Peterson, Brian; SMALL, BRIAN - Southern Illinois University

Submitted to: Aquaculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/2/2016
Publication Date: 6/1/2016
Citation: Schroeter, J., Peterson, B.C., Small, B.C. 2016. Development of multitissue microfluidic dynamic array for assessing changes in gene expression associated with channel catfish appetite, growth, metabolism, and intestinal health. Aquaculture. 464: 213-221.

Interpretive Summary: Large-scale, gene expression methods allow for high throughput analysis of physiological pathways at a fraction of the cost of individual gene expression analysis. Systems, such as the Fluidigm quantitative PCR array described here, can provide powerful assessments of the effects of diet, environment, and management on physiological pathways affecting production parameters. Genes measured with the microfluidic array used in this study were chosen to represent key pathways involved in appetite, growth, metabolism, and intestinal inflammation for their potential to provide insight into the effects of diet and dietary supplements on these important physiological processes regulating feed efficiency and growth. With few exceptions, PCR primers were able to be designed from Ictaluridae gene sequences published in GenBank. All target gene primers were initially validated via real-time PCR. Combined hypothalamus/pituitary, liver, stomach, and intestine were dissected from 9 fish to validate the Fluidigm quantitative PCR array. Development of this multitissue microfluidic dynamic array paves the way for its use as a tool in the evaluation of regulatory pathways in response to alternative feeding strategies, dietary formulations, and supplementation.

Technical Abstract: Large-scale, gene expression methods allow for high throughput analysis of physiological pathways at a fraction of the cost of individual gene expression analysis. Systems, such as the Fluidigm quantitative PCR array described here, can provide powerful assessments of the effects of diet, environment, and management on physiological pathways affecting production parameters. Genes measured with the microfluidic array used in this study were chosen to represent key pathways involved in appetite, growth, metabolism, and intestinal inflammation for their potential to provide insight into the effects of diet and dietary supplements on these important physiological processes regulating feed efficiency and growth. With few exceptions, PCR primers were able to be designed from Ictaluridae gene sequences published in GenBank. PCR amplicons from primers designed outside of Ictaluridae were sequenced to verify gene identity. All target gene primers were initially validated via real-time PCR. Combined hypothalamus/pituitary, liver, stomach, and intestine were dissected from 9 fish to validate the Fluidigm quantitative PCR array and analyze the expression of 45 genes. Use of the Fluidigm system resulted in reliable cycle threshold levels (Ct), efficiencies (E), and quality scores (QS) for all genes examined except 18 S ribosomal RNA (18S), pro-opiomelanocortin (POMC), vasoactive intestinal peptide (VIP), somatostatin receptor (SSR), pepsinogen C (PEPC), cholecystokinin b (CCKb), transforming growth factor, beta 2 (TGF-B2), and melanocortin 4 receptor (MCR4). Of all potential reference genes included in the panel, alpha-tubulin (TUBA) had a high QS, E, and acceptable Ct. Development of this multitissue microfluidic dynamic array paves the way for its use as a tool in the evaluation of regulatory pathways in response to alternative feeding strategies, dietary formulations, and supplementation.