Hostname: page-component-78c5997874-m6dg7 Total loading time: 0 Render date: 2024-11-15T21:15:32.319Z Has data issue: false hasContentIssue false
  • English
  • Français

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

Published online by Cambridge University Press:  09 June 2009

Ludivine Lassois ,
Luc de Lapeyre de Bellaire  and
Haïssam Jijakli
Ludivine Lassois
Affiliation:
Gembloux Agric. Univ., Plant Pathol. Unit, Passage des Déportés 2, B-5030 Gembloux, Belgium
Luc de Lapeyre de Bellaire
Affiliation:
CIRAD, Persyst, UPR Syst. Banan. Ananas, TA B-26 / PS4, Blvd. de la Lironde, 34398 Montpellier Cedex 5, France
Haïssam Jijakli
Affiliation:
Gembloux Agric. Univ., Plant Pathol. Unit, Passage des Déportés 2, B-5030 Gembloux, Belgium
Get access

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 crown-tissue 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.

Keywords

BelgiumMusa sp.bananasfreeze-dryingRNAstorageextractionBelgiqueMusa sp.bananelyophilisationARNstockageextraction
Type
Research Article
Information
Fruits , Volume 64 , Issue 3 , May 2009 , pp. 127 - 137
Copyright
© CIRAD, EDP Sciences, 2009

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Xu, B.Y., Su, W., Liu, J.H., Wang, J.B., Jin, Z.Q., Differentially expressed cDNAs at the early stage of banana ripening identified by suppression subtractive hybridization and cDNA microarray, Planta 226 (2007) 529–539. CrossRef
Choudhury, S.R., Roy, S., Sengupta, D.N., Characterization of transcriptional profiles of MA-ACS1 and MA-ACO1 genes in response to ethylene, auxin, wounding, cold and different photoperiods during ripening in banana fruit, J. Plant Physiol. 165 (2008) 1865–1878. CrossRef
Martin L.A., Smith T.J., Obermoeller D., Bruner B., Kracklauer M., Dharmaraj S., RNA purification, in: Gerstein A.S. (Ed.), Molecular biology problem solver, Wiley-Liss, NY, USA, 2001.
Hu, C., Honda, C., Kita, M., Zhang, Z., Tsuda, T., Moriguchi, T., A simple protocol for RNA isolation from fruit trees containing high levels of polysaccharides and polyphenol compounds, Plant Mol. Biol. Rep. 20 (2002) 69a–69g. CrossRef
Ding, L., Sun, Q., Wang, Z., Sun, Y., Xu, Z., Using silica particles to isolate total RNA from plant tissues recalcitrant to extraction in guanidine thiocyanate, Anal. Biochem. 374 (2008) 426–428. CrossRef
Claros, M., Canovas, F., RNA isolation from plant tissues: a practical experience for biological undergraduates, Biochem. Educ. 27 (1999) 110–113. CrossRef
Gehrig, H., Winter, K., Cushman, J., Borland, A., Taybi, T., An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides, Plant Mol. Biol. Rep. 18 (2000) 369–376. CrossRef
Liu, W., Wang, B., Duan, C., Li, B., A method for isolating functional RNA from callus of Dendrobium candidum contented rich polysaccharides, Colloid Surf. B-Biointerfaces 42 (2005) 259–262. CrossRef
Venugopalan, C., Kapoor, H., Single step isolation of plant RNA, Phytochem. 46 (1997) 1303–1305. CrossRef
Wu, Y., Llewellyn, D., Dennis, E., A quick and easy method for isolating good-quality RNA from cotton (Gossypium hirsutum L.) tissues, Plant Mol. Biol. Rep. 20 (2002) 213–218. CrossRef
Chang, S., Puryear, J., Cairney, J., A simple and efficient method for isolating RNA from pine trees, Plant Mol. Biol. Rep. 11 (1993) 113–116. CrossRef
Gao, J., Liu, J., Li, B., Li, Z., Isolation and purification of functional total RNA from blue-grained wheat endosperm tissues containing high levels of starches and flavonoids, Plant Mol. Biol. Rep. 19 (2001) 185a–185i. CrossRef
Salzman, R.A., Fujita, T., Zhu-Salzman, K., Hasegawa, P.M., Bressan, R.A., An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates, Plant Mol. Biol. Rep. 17 (1999) 11–17. CrossRef
Zeng, Y., Yang, T., RNA isolation from highly viscous samples rich in polyphenols and polysaccharides, Plant Mol. Biol. Rep. 20 (2002) 417a–417e. CrossRef
Maniatis T., Fritsch E., Sambrook J., (Eds.). Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press, NY, USA,1982.
Chen, T., Gagliardo, R., Walker, B., Zhou, M., Shaw, C., Partial structure of the phylloxin gene from the giant monkey frog, Phyllomedusa bicolor: Parallel cloning of precursor cDNA and genomic DNA from lyophilized skin secretion, Pept. 26 (2005) 2624–2628. CrossRef
Chen, T., Xue, Y., Zhou, M., Shaw, C., Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8 a novel prokineticin from Bombina orientalis, Pept. 26 (2005) 377–383. CrossRef
Gadbois, D., Salo, W., Ann, D., Downing, S., Carlson, D., The preparation of poly(A)+mRNA from the hagfish slime gland, Prep. Biochem. Biotechnol. 18 (1988) 67–76.
Huang, Z., Ortmeyer, H., Hansen, B., Shuldiner, R., Preparation of RNA from lyophilized tissue: a stable and reliable method for long term storage, Biotech. 17 (1994) 4.
Matsuo, S., Sugiyama, T., Okuyama, T., Yoshikawa, K., Honda, K., Takahashi, R., Maeda, S., Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses, Pathol. Int. 49 (1999) 383–390. CrossRef
Tsuka H., Mori H., Okada K., Matsukawa S., Utilization of the freeze-drying method in the preparation of biologically active, intact RNA from hard frozen tissues of human prostate, Anal. Biochem. (1997) 458–461.
Vaughan, H., Chalker, V., Mee, Z., Rossouw, A., James, V., Stability of lyophilised specimens for the molecular detection of viral DNA/RNA, J. Clin. Virol. 35 (2006) 135–140. CrossRef
Sessitsch, A., Gyamfi, S., Stralis-Pavese, N., Weilharter, A., Pfeifer, U., RNA isolation from soil for bacterial community and functional analysis: evaluation of different extraction and soil conservation protocols, J. Microbiol. Methods 51 (2002) 171–179. CrossRef
Drouet, A., Hartmann, C., Polyribosomes from pear fruit, Plant Physiol. 69 (1982) 885–887. CrossRef
Jaiprakash, M., Pillai, B., Venkatesh, P., Subramanian, N., Sinkar, V., Sadhale, P., RNA isolation from high-phenolic freeze-dried tea (Camelia sinensis) leaves, Plant Mol. Biol. Rep. 21 (2003) 465a–465g. CrossRef
Saha, S., Callahan, F., Dollar, D., Creech, J., Effect of lyophilisation of cotton tissue on quality of extractable DNA, RNA, and protein, J. Cotton Sci. 1 (1997) 10–14.
Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., AFLP: a new technique for DNA fingerprinting, Nucleic Acids Res. 23 (1995) 4407–4414. CrossRef
Bachem, C., Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development, Plant J. 9 (1996) 745–753. CrossRef
Chomczynski, P., Sacchi, N., Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156–159. CrossRef
Liu, J., Goh, C., Loh, C., Liu, P., Pua, E., A method for isolation of total RNA from fruit tissues of banana, Plant Mol. Biol. Rep. 16 (1998) 1–6.
Mbéguié-A-Mbéguié, D., Fils-Lycaon, B., Chillet, M., Hubert, O., Galas, C., Gomez, R.-M., Extraction and purification of total RNA from banana tissues (small scale), Fruits 63 (4) (2008) 255–261. CrossRef
Wan, C., Wilkins, T., A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.), Anal. Biochem. 233 (1994) 7–12. CrossRef
Asif, M.H., Dhawan, P., Nath, P., A simple procedure for isolation of high quality RNA from ripening banana fruit, Plant Mol. Biol. Rep. 18 (2000) 109–115. CrossRef
Huberman, J.A., Importance of measuring nucleic acid absorbance at 240 nm as well as at 260 and 280 nm, Biotech. 18 (1995) 636.
Troutman T., Prasauckas K., Kennedy M., Stevens J., Davies M., Dadd A., How to properly use and maintain laboratory equipment, in: Gerstein A.S. (Ed.), Molecular biology problem solver, Wiley-Liss, NY, USA, 2001.
Mbeguie-A-Mbeguie, D., Hubert, O., Sabau, X., Chillet, M., Fils-Lycaon, B., Baurens, F.C., Use of suppression subtractive hybridization approach to identify genes differentially expressed during early banana fruit development undergoing changes in ethylene responsiveness, Plant Sci. 172 (2007) 1025–1036. CrossRef
Kuhn, E., From library screening to microarray technology: strategies to determine gene expression profiles and to identify differentially regulated genes in plants, Ann. Bot. 87 (2001) 139–155. CrossRef
Matz, M., Lukyanov, S.A., Different strategies of differential display: areas of application, Nucleic Acids Res. 26 (1998) 5537–5543. CrossRef
Campalans, A., Pagès, M., Messeguer, R., Identification of differentially expressed genes by the cDNA-AFLP technique during dehydration of almond, Tree Physiol. 21 (2001) 633–643. CrossRef
Brugmans, B., Fernadez del Carmen, A., Bachem, C., van Hos, H., van Eck, H.J., Visser, R., A novel method for the construction of genome wide transcriptome maps, Plant J. 31 (2002) 1–15. CrossRef