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Text
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URL Address
<a href="http://doi.org/10.1371/journal.pone.0054277" target="_blank" rel="noreferrer noopener">http://doi.org/10.1371/journal.pone.0054277</a>
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Pages
11-11
Issue
1
Volume
8
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Title
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Leptin in Whales: Validation and Measurement of mRNA Expression by Absolute Quantitative Real-Time PCR
Publisher
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PLOS ONE
Date
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2013
2013-01
Subject
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obesity; mammals; Science & Technology - Other Topics; resistance; mass; weight; quantification; housekeeping genes; reference genes; rt-pcr; serum leptin
Creator
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Ball H C; Holmes R K; Londraville R L; Thewissen J G M; Duff R J
Description
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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.
Identifier
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<a href="http://doi.org/10.1371/journal.pone.0054277" target="_blank" rel="noreferrer noopener">10.1371/journal.pone.0054277</a>
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Journal Article or Conference Abstract Publication
2013
Ball H C
Duff R J
Holmes R K
housekeeping genes
Journal Article or Conference Abstract Publication
Londraville R L
Mammals
mass
Obesity
PloS one
quantification
reference genes
resistance
rt-pcr
Science & Technology - Other Topics
serum leptin
Thewissen J G M
weight