Combining an original method for preserving RNA expressionin situwith an effective RNA extraction method makes it possible to study gene expression in any banana fruit tissue

Abstract

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...

A Combined RNA Preservation and Extraction Protocol for Gene Expression Studies in Cacao Beans

“…FD is used for a wide range of applications within the food industry, pharmacy, and biotechnology. It has been used successfully for RNA preservation in grapevine buds ( García-baldenegro et al., 2015 ), banana tissue ( Lassois et al., 2009 ), and tea leaves ( Jaiprakash et al., 2003 ). However, complete RNA degradation has been observed in FD cotton, indicating the tissue- and species-specificity of the technique ( Pearson et al., 2006 ).…”

“…An alternative is freeze drying (FD), which has been considered as a cost-effective method for temporary RNA preservation and short-term shipment of genetic sensitive samples at room temperature (RT). This method has already been successfully applied on, for instance, grapes ( García-baldenegro et al., 2015 ), bananas ( Lassois et al., 2009 ), and tea leaves ( Jaiprakash et al., 2003 ).…”

Identification of Genes Involved in the Response of Banana to Crown Rot Disease

“…The sensitivity and specificity of cDNA-AFLP analysis are comparable with those of a microarray approach (Reijans et al 2003). Its reproducibility has also been proven (Kuhn 2001;Lassois et al 2009). …”

“…One of these three was collected for crown sampling at 13 dpi when symptoms are developed (13 dpi = clusters B, D, F, and H according to the treatment and the biological replicate; ethrel treated). The crowns collected at each period (1 hbi and 13 dpi) for molecular analyses were immediately frozen in liquid nitrogen, freeze dried at -80°C for 24 h (Telstar, Cryodos, Barcelona, Spain), and stored at room temperature before RNA extraction (Lassois et al 2009) and molecular analysis.…”