Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

Many studies have shown that risk factors that are independent of blood pressure (BP) can contribute to the development of cardiac hypertrophy (CH). Among these factors, high-salt (HS) intake was prominent. Although some studies have attempted to elucidate the role of salt in the development of this disease, the mechanisms by which salt acts are not yet fully understood. Thus, the aim of this study was to better understand the mechanisms of CH and interstitial fibrosis (IF) caused by HS intake. Male Wistar rats were divided into 5 groups according to diet [normal salt (NS; 1.27% NaCl) or HS (8% NaCl)] and treatment [losartan (LOS) (HS+LOS group), hydralazine (HZ) (HS+HZ group), or N-acetylcysteine (NAC) (HS+NAC group)], which was given in the drinking water. Tail-cuff BP, transverse diameter of the cardiomyocyte, IF, angiotensin II type 1 receptor (AT1) gene and protein expression, serum aldosterone, cardiac angiotensin II, cardiac thiobarbituric acid-reactive substances, and binding of conformation-specific anti-AT1 and anti-angiotensin II type 2 receptor (AT2) antibodies in the 2 ventricles were measured. Based on the left ventricle transverse diameter data, the primary finding was the occurrence of significant BP-independent CH in the HS+HZ group (96% of the HS group) and a partial or total prevention of such hypertrophy via treatment with NAC or LOS (81% and 67% of the HS group, respectively). The significant total or partial prevention of IF using all 3 treatments (HS+HZ, 27%; HS+LOS, 27%; and HS+NAC, 58% of the HS group, respectively), and an increase in the AT1 gene and protein expression and activity in groups that developed CH, confirmed that CH occurred via the AT1 in this experimental model. Thus, this study unveiled some relevant previously unknown mechanisms of CH induced by chronic HS intake in Wistar rats. The link of oxidative stress with CH in our experimental model is very interesting and stimulates further evaluation for its full comprehension.

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“…Gene expression was assessed via RT-PCR as previously described (28). The primers used for the RT-PCR are included in Supplemental Table 1.…”

Section: Methodsmentioning

confidence: 99%

High-Salt Intake Induces Cardiomyocyte Hypertrophy in Rats in Response to Local Angiotensin II Type 1 Receptor Activation

Katayama

Pereira

Dopona

et al. 2014

The Journal of Nutrition

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“…However, this result may not be universally applied to other analyses of muscle samples, such as western blotting and enzyme activity assays, as well as to all mishandling actions. For example, the manipulations we performed with the muscle samples took place over only a short time-span, and it has been reported that RNA is not viable in tissues that have been defrosted for more than 24 hours [ 21 ]. Thus, despite our results, researchers should still be cautious with sample acquisition and handling (see our recommendations in Box 1 ).…”

Section: Step 3: Rna Assessmentmentioning

confidence: 99%

“…It has previously been reported that isolated total RNA is preserved after thawing for 24 hours at room temperature (e.g., in the case of freezer breakdown) [ 21 ]. We also introduced other inadvisable actions that could potentially introduce RNase to the sample, but these failed to degrade the RNA samples.…”

Section: Step 3: Rna Assessmentmentioning

confidence: 99%

An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research

et al. 2018

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Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experimental protocols and data analyses from different laboratories, and there is a lack of consistency of proper quality control steps throughout the assay. In this study, we present a number of experiments on various steps of quantitative PCR workflow, and demonstrate how to perform a quantitative PCR experiment with human skeletal muscle samples in an exercise study. We also tested some common mistakes in performing qPCR. Interestingly, we found that mishandling of muscle for a short time span (10 mins) before RNA extraction did not affect RNA quality, and isolated total RNA was preserved for up to one week at room temperature. Demonstrated by our data, use of unstable reference genes lead to substantial differences in the final results. Alternatively, cDNA content can be used for data normalisation; however, complete removal of RNA from cDNA samples is essential for obtaining accurate cDNA content.

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“…The gene expression was assessed via RT-PCR as previously described (28). The primers used for RT-PCR are included in Table 1.…”

Section: Rna Isolation and Gene Expression Of Ras Components In Both mentioning

confidence: 99%

Hipertrofia miocárdica induzida por consumo elevado de sal na dieta: avaliação do sistema renina-angiotensina e do efeito da N-acetilcisteína

Katayama¹

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a quem tenho como exemplo e que admiro pelo seu caráter, incentivo e inteligência ímpar. Obrigada pela oportunidade, pela confiança e por todos estes anos de aprendizado. O senhor foi fundamental para minha formação profissional e pessoal. Dra. Luzia Naoko Shinohara Furukawa pela disposição em ajudar sempre, pelos ensinamentos e amizade. Serei eternamente grata e sentirei muitas saudades! A Ivone Braga de Oliveira pela ajuda diária, paciência e amizade. Ainda volto para gente tomar as nossas cervejas! Vou sentir sua falta! Dra. Miriam Sterman Dolnikoff pelo apoio profissional e pessoal. À querida Helô Shimizu pelas dosagens de TBARS e pelas piadas e risadas nos corredores. À amiga Dra. Karen L. L. Jang por estar ao meu lado durante todos esses anos de laboratório, pelas risadas e viagens. Você é um exemplo de competência! À amiga Sônia de Fátima Soto pela amizade, ajuda e companheirismo. Obrigada por dividir todo o estresse do Western Blot comigo, você foi muito importante nesse processo. Sentirei falta de dividir o quarto com você nas viagens! Ao Rafael Canavel Pereira por toda ajuda com os animais e em todo projeto. Obrigada pela oportunidade de me ensinar a orientar. Levarei sua amizade para sempre com carinho. À Ellen P. de Brito Dopona por toda ajuda no projeto. Sua contribuição para o projeto e para mim foram extremamentes importantes. À Daniele Nunes Ferreira, a quem devo todo meu aprendizado como aluna de iniciação científica. Você me ensinou muito e contribuiu para que eu me tornasse mais crítica e independente. Obrigada! À Carolina Romão e Guilherme Marchesi pela amizade, pelas brincadeiras e risadas. O ambiente de trabalho ficava muito mais divertido com vocês. À Dra. Camilla F. Wenceslau que passou um curto período conosco no laboratório, mas trouxe ensinamentos fundamentais para esse projeto e se tornou uma querida amiga. À Dra. Débora Rothstein Ramos, pela amizade construída nos últimos anos e pela ajuda que sempre me deu. À todos os amigos do Laboratório de Hipertensão Experimental, Flávia, Priscila, Carina e Maria Angélica pelo apoio e amizade. Ao Prof. Dr. Rui de Toledo Barros e ao Prof. Dr. Roberto Zatz por terem me aceitado no programa de pós-graduação da Nefrologia. Ao Walter Campestre pelos cuidados com os animais, mas principalmente pelas risadas e amizade. À todos do LIM 16 e da FMUSP que de alguma forma direta ou indiretamente, contribuíram para a realização deste trabalho e para minha formação. Aos meus pais Paulo e Bartira, pelo amor incondicional, pelas lutas incessantes pelo meu melhor, por serem meus maiores exemplos de vida e por sempre me guiarem pelo caminho do bem. Amo muito vocês! Aos meus irmãos Juninho, Thata e Analu por serem meus primeiros companheiros de vida. Obrigada pelo forte sentimento que nos une e que só a gente é capaz de entender. Amo muito vocês! Aos meus cunhados Ana Paula e Miller por serem os irmãos que a vida me trouxe e principalmente por completarem nossa família e fazerem meus irmãos felizes. Amo vocês! Aos meus sobrinhos Luca e Duda por serem a luz e alegri...

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Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

Cited by 6 publications

References 9 publications

High-Salt Intake Induces Cardiomyocyte Hypertrophy in Rats in Response to Local Angiotensin II Type 1 Receptor Activation

High-Salt Intake Induces Cardiomyocyte Hypertrophy in Rats in Response to Local Angiotensin II Type 1 Receptor Activation

An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research

Hipertrofia miocárdica induzida por consumo elevado de sal na dieta: avaliação do sistema renina-angiotensina e do efeito da N-acetilcisteína

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