Defining Suitable Reference Genes for RT-qPCR Analysis on Intestinal Epithelial Cells

Sirakov, M; Borra, Marco; Cambuli, Francesca Maria; Plateroti, Michelina

doi:10.1007/s12033-012-9643-3

Cited by 11 publications

(11 citation statements)

References 41 publications

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“…In the case of miRNA normalization, the evaluated candidate RGs included miR-16, miR-19a, miR-122, miR-142, miR-143, miR-186, miR-200a, small nucleolar RNA 202 and 234 (sno202, sno234) and small nuclear RNA U6 (U6). All candidate RGs for mRNA and miRNA normalization were selected considering 1) that they belong to different functional classes in order to reduce the chance of co-regulation of their genes, 2) their common use as endogenous controls and 3) their relative quantities in liver and/or SI [29]–[33].…”

Section: Resultsmentioning

confidence: 99%

Reference Genes for Real-Time PCR Quantification of Messenger RNAs and MicroRNAs in Mouse Model of Obesity

et al. 2014

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Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of NAD(P)H:quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice.

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Section: Resultsmentioning

confidence: 99%

Reference Genes for Real-Time PCR Quantification of Messenger RNAs and MicroRNAs in Mouse Model of Obesity

et al. 2014

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“…cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (#4368813, Applied Biosystems) and used for real time quantitative PCR (RT-qPCR) reactions using FAST SYBR green and the Viia7 Real Time PCR system (Applied Biosystems). Each sample was run in triplicate and the best duplicates or triplicates were analyzed using the 2 −∆∆C T method 25 with Ppib as the reference gene 26 . Primers used are listed in Sup Table 1 .…”

Section: Methodsmentioning

confidence: 99%

Enhancing enterocyte fatty acid oxidation in mice affects glycemic control depending on dietary fat

Ramachandran

Clara

Fedele

et al. 2018

Sci Rep

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Studies indicate that modulating enterocyte metabolism might affect whole body glucose homeostasis and the development of diet-induced obesity (DIO). We tested whether enhancing enterocyte fatty acid oxidation (FAO) could protect mice from DIO and impaired glycemic control. To this end, we used mice expressing a mutant form of carnitine palmitoyltransferase-1a (CPT1mt), insensitive to inhibition by malonyl-CoA, in their enterocytes (iCPT1mt) and fed them low-fat control diet (CD) or high-fat diet (HFD) chronically. CPT1mt expression led to an upregulation of FAO in the enterocytes. On CD, iCPT1mt mice had impaired glycemic control and showed concomitant activation of lipogenesis, glycolysis and gluconeogenesis in their enterocytes. On HFD, both iCPT1mt and control mice developed DIO, but iCPT1mt mice showed improved glycemic control and reduced visceral fat mass. Together these data indicate that modulating enterocyte metabolism in iCPT1mt mice affects glycemic control in a body weight-independent, but dietary fat-dependent manner.

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“…cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems #4368813) and used for RT-qPCR reactions using FAST SYBR green and the Viia7 Real Time PCR system (Applied Biosystems). Each sample was run in triplicate and analysed using the 2 −ΔΔC T method [27] with Peptidylprolyl Isomerase B ( Ppib ) as the reference gene [28] . Primers used are listed in Sup Table 2 .…”

Section: Methodsmentioning

confidence: 99%

Intestinal SIRT3 overexpression in mice improves whole body glucose homeostasis independent of body weight

Ramachandran

Clara

Fedele

et al. 2017

Molecular Metabolism

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ObjectiveIntestinal metabolism might play a greater role in regulating whole body metabolism than previously believed. We aimed to enhance enterocyte metabolism in mice and investigate if it plays a role in diet-induced obesity (DIO) and its comorbidities.MethodsUsing the cre-loxP system, we overexpressed the mitochondrial NAD+ dependent protein deacetylase SIRT3 in enterocytes of mice (iSIRT3 mice). We chronically fed iSIRT3 mice and floxed-SIRT3 control (S3fl) mice a low-fat, control diet (CD) or a high-fat diet (HFD) and then phenotyped the mice.ResultsThere were no genotype differences in any of the parameters tested when the mice were fed CD. Also, iSIRT3 mice were equally susceptible to the development of DIO as S3fl mice when fed HFD. They were, however, better able than S3fl mice to regulate their blood glucose levels in response to exogenous insulin and glucose, indicating that they were protected from developing insulin resistance. This improved glucose homeostasis was accompanied by an increase in enterocyte metabolic activity and an upregulation of ketogenic gene expression in the small intestine.ConclusionEnhancing enterocyte oxidative metabolism can improve whole body glucose homeostasis.

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Defining Suitable Reference Genes for RT-qPCR Analysis on Intestinal Epithelial Cells

Cited by 11 publications

References 41 publications

Reference Genes for Real-Time PCR Quantification of Messenger RNAs and MicroRNAs in Mouse Model of Obesity

Reference Genes for Real-Time PCR Quantification of Messenger RNAs and MicroRNAs in Mouse Model of Obesity

Enhancing enterocyte fatty acid oxidation in mice affects glycemic control depending on dietary fat

Intestinal SIRT3 overexpression in mice improves whole body glucose homeostasis independent of body weight

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