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      RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

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          Abstract

          Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

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          Reference genes in real-time PCR

          This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition.
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            The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization.

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              Insulin resistance (HOMA-IR) cut-off values and the metabolic syndrome in a general adult population: effect of gender and age: EPIRCE cross-sectional study

              Background Insulin resistance has been associated with metabolic and hemodynamic alterations and higher cardio metabolic risk. There is great variability in the threshold homeostasis model assessment of insulin resistance (HOMA-IR) levels to define insulin resistance. The purpose of this study was to describe the influence of age and gender in the estimation of HOMA-IR optimal cut-off values to identify subjects with higher cardio metabolic risk in a general adult population. Methods It included 2459 adults (range 20–92 years, 58.4% women) in a random Spanish population sample. As an accurate indicator of cardio metabolic risk, Metabolic Syndrome (MetS), both by International Diabetes Federation criteria and by Adult Treatment Panel III criteria, were used. The effect of age was analyzed in individuals with and without diabetes mellitus separately. ROC regression methodology was used to evaluate the effect of age on HOMA-IR performance in classifying cardio metabolic risk. Results In Spanish population the threshold value of HOMA-IR drops from 3.46 using 90th percentile criteria to 2.05 taking into account of MetS components. In non-diabetic women, but no in men, we found a significant non-linear effect of age on the accuracy of HOMA-IR. In non-diabetic men, the cut-off values were 1.85. All values are between 70th-75th percentiles of HOMA-IR levels in adult Spanish population. Conclusions The consideration of the cardio metabolic risk to establish the cut-off points of HOMA-IR, to define insulin resistance instead of using a percentile of the population distribution, would increase its clinical utility in identifying those patients in whom the presence of multiple metabolic risk factors imparts an increased metabolic and cardiovascular risk. The threshold levels must be modified by age in non-diabetic women.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                15 June 2016
                2016
                : 11
                : 6
                : e0157002
                Affiliations
                [1 ]IBIMA, Universidad de Málaga, Campus Teatinos s/n, 29010, Málaga, España
                [2 ]CIBER Pathophysiology of obesity and nutrition CB06/03, Carlos III Health Institute, Malaga, 29010, Spain, Laboratory of Biomedical Research, Virgen de la Victoria Clinical University Hospital, Málaga, 29010, Spain
                [3 ]Biomedical Sciences Program, Health Sciences Department, College of Arts and Sciences, Qatar University, P.O. Box 2713, Doha, Qatar
                [4 ]Unidad de Gestión Clínica de Alergia Intercentros, Hospital Universitario Virgen Macarena, Sevilla, Spain
                [5 ]Unidad de Gestion Clínica Intercentros de Endocrinología y Nutrición, Instituto de Investigación Biomédica de Málaga (IBIMA), Hospital Regional Universitario de Málaga/Universidad de Málaga, 29009, Málaga, Spain
                [6 ]Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Málaga, Spain
                [7 ]Endocrinology Service, Virgen de la Victoria Clinical University Hospital, Malaga, 29010, Spain
                Naval Research Laboratory, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: RE. Performed the experiments: AMG SL LCA WOO HZ JM SYRZ. Analyzed the data: RE FJBS AMG AVR JM SYRZ. Wrote the paper: RE FJBS AMG AVR. Recruited and carried out the surgeries: FJT.

                Article
                PONE-D-15-46947
                10.1371/journal.pone.0157002
                4909211
                27304673
                f63e6345-bb53-44db-9f4f-361e17b24a92
                © 2016 Gentile et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 26 October 2015
                : 23 May 2016
                Page count
                Figures: 4, Tables: 6, Pages: 18
                Funding
                Funded by: from the Spanish Ministry of Health and co-funded by Fondo Europeo de Desarrollo Regional–FEDER
                Award ID: PI10/01947, PI13/02628
                Award Recipient :
                Funded by: Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía and co-funded by Fondo Europeo de Desarrollo Regional–FEDER
                Award ID: CTS-7895
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004587, Instituto de Salud Carlos III;
                Award ID: El Bekay is under a contract Miguel Servet type II (CPII13/00041)
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004587, Instituto de Salud Carlos III;
                Award ID: FJBS is a recipient of a "Miguel Servet II" research contract (CPII13/00042)
                Award Recipient :
                Funded by: Consejeria de salud
                Award ID: FJBS is a recipient of a "Miguel Servet II" research contract (CPII13/00042) and also belongs to the regional "Nicolás Monardes" research program of the Consejería de Salud (C-0070-2012; Junta de Andalucía, Spain)
                Funded by: FIS-Thematic Networks and Co-Operative Research Centres RIRAAF
                Award ID: (RD07-0064)
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003329, Ministerio de Economía y Competitividad;
                Award ID: AV-R is under a contract Proyectos de I+D+i para jóvenes investigadores (SAF2014-60649-JIN)
                Award Recipient :
                This work was supported by grants from the Instituto de Salud Carlos III (PI10/01947, PI13/02628) with Fondos FEDER and the Consejería de Economía e Innovación, Ciencia y Empleo, Junta de Andalucía (CTS-7895) with Fondos FEDER. R. El Bekay is under a contract Miguel Servet type II (CPII13/00041) from the Instituto de Salud Carlos III. F-JB-S is a recipient of a "Miguel Servet II" research contract (CPII13/00042) and also belongs to the regional "Nicolás Monardes" research program of the Consejería de Salud (C-0070-2012; Junta de Andalucía, Spain). This work was supported by the FIS-Thematic Networks and Co-Operative Research Centres RIRAAF (RD07-0064). JM is under the Programa de Intensificación de la Actividad Investigadora del Sistema Nacional de Salud. AV-R is under a contract Proyectos de I+D+i para jóvenes investigadores from the Ministerio de Economía y Competitividad (SAF2014-60649-JIN). S-YR-Z is recipient of a post-doctoral contract from Consejería de Salud de la Junta de Andalucía (RH-0070-2013).
                Categories
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                Medicine and Health Sciences
                Endocrinology
                Endocrine Physiology
                Insulin Resistance
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                Endocrine Physiology
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