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Leptin in whales: validation and measurement of mRNA expression by absolute quantitative real-time PCR.

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.

SUBMITTER: Ball HC

PROVIDER: S-EPMC3546977 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis-halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted.ResultsTen candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae.ConclusionsOverall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A. littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed.

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Leptin in whales: validation and measurement of mRNA expression by absolute quantitative real-time PCR.

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