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Combining an original method for preserving RNA expression in situ with an effective RNA extraction method makes it possible to study gene expression in any banana fruit tissue. Abstract-Introduction. RNA isolation is a prerequisite to studying gene expression in banana and to understanding changes occurring in response to the environment. Standard extraction methods do not efficiently extract RNA from plants such as banana, with high levels of phenolics, carbohydrates, or other compounds that bind to and/or coprecipitate with RNA. Materials and methods. Five to seven RNA extraction methods were compared. Four crowntissue storage methods were also compared. cDNA-AFLP was used to ensure that the obtained RNA was of sufficient quality for molecular applications and that RNA expression was unaltered by in situ storage. Results and discussion. The modified hot-borate method proved to be the best RNA extraction method, allowing high yields of good quality, undegraded RNA from the crown, fruit peel and pulp at all stages of ripening. The RNA obtained by this method was of sufficient quality for molecular applications such as cDNA-AFLP that give highly reproducible results. Freeze-drying of fresh tissues and tissue conservation in hot-borate buffer, two original storage methods, appear appropriate for preserving RNA in situ without ultra-low temperature. The RNA obtained was of high quality, undegraded, and useful for all downstream applications. The genome expression profile obtained by cDNA-AFLP analysis was unaltered by these methods for storing collected tissues. Conclusion. By applying all the suggested procedures in this work, it is possible to store and study gene expression in any banana fruit tissue, whatever the maturity stage, without affecting the RNA expression level. Belgium / Musa sp. / bananas / freeze-drying / RNA / storage / extraction La combinaison d'une méthode originale permettant de maintenir l'expression de l'ARN in situ et d'une méthode efficace pour extraire l'ARN permet d'étudier l'expression de gènes dans n'importe quel tissu de banane. Résumé-Introduction. L'extraction de l'ARN est un préalable pour étudier l'expression des gènes dans la banane et pour comprendre les changements intervenant en réponse à l'environnement. Les méthodes standard d'extraction de l'ARN ne sont pas efficaces avec des plantes comme le bananier, qui présentent des niveaux élevés de composés phénoliques, d'hydrates de carbone, ou autres composés qui se lient à et/ou coprécipitent l'ARN. Matériel et méthodes. Cinq à sept méthodes d'extraction de l'ARN ont été comparées. Quatre méthodes de stockage de tissus de couronne ont été également comparées. La technique de cDNA-AFLP a été utilisée pour s'assurer que l'ARN extrait était de qualité suffisante pour des applications moléculaires et que l'expression de l'ARN était inchangée après un stockage in situ. Résultats et discussion. La méthode du borate chaud modifiée s'est avérée être la meilleure méthode d'extraction de l'ARN, permettant des rendements élevés de bonne qual...
Combining an original method for preserving RNA expression in situ with an effective RNA extraction method makes it possible to study gene expression in any banana fruit tissue. Abstract-Introduction. RNA isolation is a prerequisite to studying gene expression in banana and to understanding changes occurring in response to the environment. Standard extraction methods do not efficiently extract RNA from plants such as banana, with high levels of phenolics, carbohydrates, or other compounds that bind to and/or coprecipitate with RNA. Materials and methods. Five to seven RNA extraction methods were compared. Four crowntissue storage methods were also compared. cDNA-AFLP was used to ensure that the obtained RNA was of sufficient quality for molecular applications and that RNA expression was unaltered by in situ storage. Results and discussion. The modified hot-borate method proved to be the best RNA extraction method, allowing high yields of good quality, undegraded RNA from the crown, fruit peel and pulp at all stages of ripening. The RNA obtained by this method was of sufficient quality for molecular applications such as cDNA-AFLP that give highly reproducible results. Freeze-drying of fresh tissues and tissue conservation in hot-borate buffer, two original storage methods, appear appropriate for preserving RNA in situ without ultra-low temperature. The RNA obtained was of high quality, undegraded, and useful for all downstream applications. The genome expression profile obtained by cDNA-AFLP analysis was unaltered by these methods for storing collected tissues. Conclusion. By applying all the suggested procedures in this work, it is possible to store and study gene expression in any banana fruit tissue, whatever the maturity stage, without affecting the RNA expression level. Belgium / Musa sp. / bananas / freeze-drying / RNA / storage / extraction La combinaison d'une méthode originale permettant de maintenir l'expression de l'ARN in situ et d'une méthode efficace pour extraire l'ARN permet d'étudier l'expression de gènes dans n'importe quel tissu de banane. Résumé-Introduction. L'extraction de l'ARN est un préalable pour étudier l'expression des gènes dans la banane et pour comprendre les changements intervenant en réponse à l'environnement. Les méthodes standard d'extraction de l'ARN ne sont pas efficaces avec des plantes comme le bananier, qui présentent des niveaux élevés de composés phénoliques, d'hydrates de carbone, ou autres composés qui se lient à et/ou coprécipitent l'ARN. Matériel et méthodes. Cinq à sept méthodes d'extraction de l'ARN ont été comparées. Quatre méthodes de stockage de tissus de couronne ont été également comparées. La technique de cDNA-AFLP a été utilisée pour s'assurer que l'ARN extrait était de qualité suffisante pour des applications moléculaires et que l'expression de l'ARN était inchangée après un stockage in situ. Résultats et discussion. La méthode du borate chaud modifiée s'est avérée être la meilleure méthode d'extraction de l'ARN, permettant des rendements élevés de bonne qual...
Reverse transcription and quantitative polymerase chain reaction (RT-qPCR) has become the most common method for characterizing expression patterns of individual mRNAs due to a large dynamic range of linear quantification, high speed, sensitivity, resolution and cost-effectiveness. However, the complexity of the protocol, variability of reagents, an inconsistent quality of biological samples, and the absence of standardized methods of data quantification may produce inconsistent results. In an effort to to standardize ithe procedure and assure high reliability of data, the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines was defined and further extended by Prof. Bustin and colleagues (2004). These guidelines have been followed by the development of an XML-based real-time PCR data markup language (RDML) and a RDML data base for consistent reporting of RT-qPCR data. Here we follow the RT-qPCR procedure step by step in compliance with MIQE requirements, local facilities and resources and our own experience in application of RT-qPCR methodology. K e y w o r d s: RT-qPCR, normalization and standardization of data, MIQE "Garbage in, garbage out" a teaching mantra by George Fuechsel, an IBM 305 RAMAC technician/instructor, N.Y. 50s XX c. (Early programmers were required to test virtually each program step)
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