Combining an original method for preserving RNA expression in situ with an effetive RNA method makes it possible to study gene expression in any banana fruit tissue.
Lassois, Ludivine; de Lapeyre de Bellaire, Luc; Jijakli, Haissam
[en] 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. [fr] 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 qualité, et donnant de l'ARN non dégradé
à partir des tissus de la couronne, de la peau des fruits et de la pulpe à toutes les étapes de la
maturation. L'ARN obtenu par cette méthode a été de qualité suffisante pour les applications
moléculaires telles que le cDNA-AFLP qui donne des résultats fortement reproductibles. La lyophilisation
des tissus frais et la conservation de tissu dans la solution tampon de borate chaud,
deux méthodes originales de stockage, semblent appropriées à la conservation de l'ARN in situ
en absence d’ultra basse température. L'ARN obtenu a été de qualité, non dégradé, et utilisable
pour toutes les applications. Le profil d'expression du génome obtenu par la technique de cDNAAFLP
est resté inchangé par ces méthodes de stockage des tissus collectés. Conclusion. En
appliquant toutes les procédures suggérées dans ce travail, il est possible de stocker et d’étudier
l'expression de gènes dans n'importe quel tissu de banane, quel que soit le stade de maturité,
sans affecter le niveau d'expression de l'ARN.
Combining an original method for preserving RNA expression in situ with an effetive RNA method makes it possible to study gene expression in any banana fruit tissue.
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