Abstract
Leptin is the primary hormone in mammals that regulates adipose stores. Arctic adapted cetaceans maintain enormous adipose depots, suggesting possible modifications of leptin or receptor function. Determining expression of these genes is the first step to understanding the extreme physiology of these animals, and the uniqueness of these animals presents special challenges in estimating and comparing expression levels of mRNA transcripts. Here, we compare expression of two model genes, leptin and leptin-receptor gene-related product (OB-RGRP), using two quantitative real-time PCR (qPCR) methods: “relative” and “absolute”. To assess the expression of leptin and OB-RGRP in cetacean tissues, we first examined how relative expression of those genes might differ when normalized to four common endogenous control genes. We performed relative expression qPCR assays measuring the amplification of these two model target genes relative to amplification of 18S ribosomal RNA (18S), ubiquitously expressed transcript (Uxt), ribosomal protein 9 (Rs9) and ribosomal protein 15 (Rs15) endogenous controls. Results demonstrated significant differences in the expression of both genes when different control genes were employed; emphasizing a limitation of relative qPCR assays, especially in studies where differences in physiology and/or a lack of knowledge regarding levels and patterns of expression of common control genes may possibly affect data interpretation. To validate the absolute quantitative qPCR methods, we evaluated the effects of plasmid structure, the purity of the plasmid standard preparation and the influence of type of qPCR “background” material on qPCR amplification efficiencies and copy number determination of both model genes, in multiple tissues from one male bowhead whale. Results indicate that linear plasmids are more reliable than circular plasmid standards, no significant differences in copy number estimation based upon background material used, and that the use of ethanol precipitated, linearized plasmid preparation produce the most reliable results.
Highlights
Leptin is a small, 16 kDa peptide hormone encoded by the obese gene in vertebrates [1]
To validate the absolute quantitative quantitative real-time PCR (qPCR) methods used to quantify cetacean leptin and OB-RGRP gene expression we evaluated the effects of plasmid structure, the purity of the plasmid standard preparation and the influence of type of qPCR ‘‘background’’ material (RNA or cDNA) on qPCR amplification efficiencies and copy number determination of both model genes in various tissues from one male bowhead whale (09B9). qPCR amplification efficiencies and any significant differences observed in threshold cycle (Ct) values and copy numbers were determined and compared for each test and each gene
Do these large adipose stores correlate to high leptin mRNA expression in these species? Relative qPCR assays would address this question among individuals and tissues on a single plate, but only absolute quantification methods would allow us to address this question among individuals and the two different species
Summary
16 kDa peptide hormone encoded by the obese (ob) gene in vertebrates [1]. Leptin was originally characterized as a ‘‘lipostat’’ involved in regulation of body fat stores; synthesized primarily by adipose cells and secreted into the blood where it circulates to bind to receptors (obese gene receptors or OBRs) found in the hypothalamus [2]. Leptin stimulates transcription of genes up-regulating metabolic rate and lipid catabolism and down regulating appetite [2]. In this fashion, the brain monitors and balances lipid stores through the regulation of appetite and metabolism. Recent work in mice suggests that OB-RGRP regulates post-translational Golgi processing of the leptin receptor itself, regulating the number of receptor copies functional at the cell surface making it a possible mechanism of temporal and tissuespecific regulation of receptor function [4,5]
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