Dataset Information

Measurement of altered APP isoform expression in adipose tissue of diet-induced obese mice by absolute quantitative real-time PCR.

ABSTRACT: Obesity is associated with increased risk of Alzheimer's disease. Previous studies have demonstrated that amyloid-beta precursor protein (APP) is expressed in subcutaneous adipose tissue (SAT), upregulated with obesity, and correlates with insulin resistance and adipose tissue inflammation. APP is alternatively spliced into several isoforms, which may be indicative of the pathogenesis of APP-related diseases, but the accurate quantification has been difficult to standardize and reproduce. In light of this, we developed isoform-specific absolute cDNA standards for absolute quantitative real-time PCR (AQ-PCR), and measured transcript copy numbers for three major APP isoforms (APP770, APP751, and APP695), in SAT from C57BL/6 mice fed either a normal or high-fat diet. Expression of all three major APP isoforms was increased in diet-induced obese mice. Transcript copy numbers of APP770 and APP695 correlated with plasma insulin and CCL2 gene expression. The ratios of APP770 and APP751 to APP695 gradually decreased with aging, and correlated with plasma glucose levels. In addition, APP770 was significantly decreased in thiazolidinedione-treated mice. We describe quantification of APP isoform transcripts by AQ-PCR, which allows for direct comparison of gene copy number across isoforms, between experiments, and across studies conducted by independent research groups, which relative quantitative PCR does not allow. Our results suggest a possible role of differential expression of APP isoforms in the development of obesity-related insulin resistance and adipose tissue inflammation. In addition, it is important to determine if altered ratios of APP isoforms in SAT contribute to higher circulating A? peptides and increased risk of abnormalities in obesity.

SUBMITTER: Min H

PROVIDER: S-EPMC6138354 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Measurement of altered APP isoform expression in adipose tissue of diet-induced obese mice by absolute quantitative real-time PCR.

Min Hansol H Kim Jinil J Kim Young-Jin YJ Yoon Mi-Sook MS Pratley Richard E RE Lee Yong-Ho YH

Animal cells and systems 20170217 2

Obesity is associated with increased risk of Alzheimer's disease. Previous studies have demonstrated that amyloid-beta precursor protein (APP) is expressed in subcutaneous adipose tissue (SAT), upregulated with obesity, and correlates with insulin resistance and adipose tissue inflammation. APP is alternatively spliced into several isoforms, which may be indicative of the pathogenesis of APP-related diseases, but the accurate quantification has been difficult to standardize and reproduce. In lig ...[more]

PMID: 30460057

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Project description: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.

Project description:BACKGROUND: In real-time PCR data analysis, the cycle threshold (CT) method is currently the gold standard. This method is based on an assumption of equal PCR efficiency in all reactions, and precision may suffer if this condition is not met. Nonlinear regression analysis (NLR) or curve fitting has therefore been suggested as an alternative to the cycle threshold method for absolute quantitation. The advantages of NLR are that the individual sample efficiency is simulated by the model and that absolute quantitation is possible without a standard curve, releasing reaction wells for unknown samples. However, the calculation method has not been evaluated systematically and has not previously been applied to a TaqMan platform. AIM: To develop and evaluate an automated NLR algorithm capable of generating batch production regression analysis. RESULTS: Total RNA samples extracted from human gastric mucosa were reverse transcribed and analysed for TNFA, IL18 and ACTB by TaqMan real-time PCR. Fluorescence data were analysed by the regular CT method with a standard curve, and by NLR with a positive control for conversion of fluorescence intensity to copy number, and for this purpose an automated algorithm was written in SPSS syntax. Eleven separate regression models were tested, and the output data was subjected to Altman-Bland analysis. The Altman-Bland analysis showed that the best regression model yielded quantitative data with an intra-assay variation of 58% vs. 24% for the CT derived copy numbers, and with a mean inter-method deviation of x 0.8. CONCLUSION: NLR can be automated for batch production analysis, but the CT method is more precise for absolute quantitation in the present setting. The observed inter-method deviation is an indication that assessment of the fluorescence conversion factor used in the regression method can be improved. However, the versatility depends on the level of precision required, and in some settings the increased cost effectiveness of NLR may justify the lower precision.

Project description:BackgroundSince real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants.ResultsWe directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/ConclusionWe present proof of the principle that a mechanistically based model can be fit to observations from a single PCR amplification. Initial amounts of amplicon are well estimated without using a standard solution. Using the ratio of the predicted initial amounts of amplicon from 2 PCRs is shown to work well even when the absolute amounts of amplicon are underestimated in the individual PCRs.

Dataset Information

Measurement of altered APP isoform expression in adipose tissue of diet-induced obese mice by absolute quantitative real-time PCR.

Publications

Measurement of altered APP isoform expression in adipose tissue of diet-induced obese mice by absolute quantitative real-time PCR.

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