ABSTRACT
qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb, Gapdh, Tbp, Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative ΔCq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16-18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha, Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh, Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development.
Subject(s)
Heart/embryology , Prosencephalon/embryology , Real-Time Polymerase Chain Reaction/standards , Somites/embryology , Animals , Gene Expression Profiling/standards , Gene Expression Regulation, Developmental , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Mice , Prosencephalon/chemistry , Reference Standards , Software , Somites/chemistry , TATA-Box Binding Protein/genetics , Tissue DistributionABSTRACT
INTRODUÇÃO: Existe uma série de fatores que favorecem o desenvolvimento de distúrbios nutricionais em crianças com paralisia cerebral, como as alterações motoras e distúrbios de deglutição. OBJETIVO: Comparar a concordância do estado nutricional de crianças e adolescentes portadores de Paralisia Cerebral por meio de curvas de crescimento. MÉTODOS: Estudo transversal realizado com 99 pacientes portadores de paralisia cerebral com idade entre 2 a 19 anos, ambos sexos, atendidos no centro de referência do nordeste, entre janeiro de 2015 a janeiro de 2017. O estado nutricional foi avaliado segundo indicadores antropométricos: peso para idade, índice de massa corporal para idade e estatura para idade tanto na curva da Organização Mundial de Saúde 2006/2007, quanto nas curvas de Paralisia cerebral. A concordância foi medida a partir do índice ponderado kappa, obteve-se um nível de significância de 5%. RESULTADO: O teste de kappa evidenciou discordância significativa entre os indicadores peso para idade, índice de massa corporal para idade e estatura para idade com seguintes valore: r= 0,008 (p= 0,001), r=0.0038 (p= 0,001) e r=0.028 (p= 0,001) respectivamente. DISCUSSÃO: No estudo visualizou-se que métodos de avaliação desenvolvidos e aprovados a partir de parâmetros de populações sadias tendem a rotular portadores de paralisia cerebral em déficit nutricional. CONCLUSÃO: O estudo sugere que a antropometria e o crescimento das crianças com paralisia cerebral divergem das crianças sadias, superestimando a desnutrição em indivíduos com paralisia cerebral
INTRODUCTION: There are a number of factors that favor the development of nutritional disorders in children with cerebral palsy, such as motor disorders and swallowing disorders. OBJECTIVE: To compare the agreement of the nutritional status of children and adolescents with cerebral palsy through growth curves. METHODS: Cross-sectional study of 99 patients with cerebral palsy aged 2 to 19 years old, both genders, treated at the northeast referral center, from January 2015 to January 2017. Nutritional status was assessed according to anthropometric indicators: weight for age, body mass index for age and height for age both on the World Health Organization curve 2006/2007 and on cerebral palsy curves. Agreement was measured from the weighted kappa index, and a significance level of 5% was obtained. RESULT: The kappa test showed significant disagreement between the indicators weight for age, body mass index for age and height for age with the following values: r = 0.008 (p = 0.001), r = 0.0038 (p = 0.001) and r = 0.028 (p = 0.001) respectively. DISCUSSION: The study showed that evaluation methods developed and approved based on healthy population parameters tend to label patients with cerebral palsy in nutritional deficits. CONCLUSION: The study suggests that anthropometry and growth in children with cerebral palsy diverge from healthy children, overestimating malnutrition in individuals with cerebral palsy
