A Method for Isolating Total RNA from Mature Buds and Other Woody Tissues of Vitis Vinifera

  • Atiako Kwame Acheampong
  • Ariel Rotman
  • Chuanlin Zheng
  • Alexandra Keren
  • Tamar Halaly
  • Omer Crane
  • Aliza Ogrodovitch
  • Etti Or
Chapter

Abstract

Toughness of plant materials and their high secondary plant metabolites, polysaccharides and proteins that bind to and/or co-precipitate with the RNA, are some of the major constraints when extracting total RNA from woody tissues such as mature buds and woody stems. Here, we detail an efficient method for isolating total RNA from woody tissues of grapes. RNA was extracted with high ionic strength buffer at 65°C. Proteins were denatured, and secondary metabolites removed by repeated phenol:chloroform:isoamyl alcohol extractions. The RNA was separated from the DNA by selective precipitation with lithium chloride (LiCl) solution. Though the procedure is laborious and time-consuming, the yield and quality of the RNA extracted were higher compared to other conventional extraction protocols. Yield and purity were spectrophotometrically monitored by UV absorbance (A260/A280 and A260/A230). The yield was about 180 μg total RNA per gram of tissue, and the A260/A280 and A260/A230 absorbance ratios were greater than 2.0. Standard reverse transcription PCR (RT-PCR) yielded 3.5 kb products from RNA isolated by this protocol. Isolated RNA has also been applied in other molecular application such as real-time PCR, Northern blot, dot blot and gene expression studies using Affymetrix Chips.

Keywords

Cetyltrimethyl Ammonium Bromide Isoamyl Alcohol Diethyl Pyrocarbonate Woody Tissue Berry Skin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

DEPC

Diethyl pyrocarbonate

CTAB

Cetyltrimethyl ammonium bromide

PVP

Polyvinylpyrrolidone

EDTA

Ethylenediaminetetraacetic acid

SDS

Sodium dodecyl sulfate

CIA

Chloroform:Isoamyl alcohol

PCIA

Phenol:Chloroform:Isoamyl alcohol

References

  1. Ainsworth C (1994) Isolation of RNA from floral tissue of Rumex acetosa (Sorrel). Plant Mol Biol Report 12:198–203.CrossRefGoogle Scholar
  2. Asif M, Trivedi P, Solomos T, Tucker M (2006) Isolation of high–quality RNA from apple (Malus domestica) fruit. J Agric Food Chem 54:5227–5229PubMedCrossRefGoogle Scholar
  3. Bilgin DD, DeLucia EA, Clough SJ (2009) A robust plant RNA isolation method suitable for Affymetrix Gene Chip analysis and quantitative real–time RT–PCR. Nature Protocols 4(3):334–340CrossRefGoogle Scholar
  4. Boss PK, Davies C, Robinson SP (1996) Analysis of the expression of anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape berries and the implications for pathway regulation. Plant Physiol 111:1059–1066PubMedGoogle Scholar
  5. Carra A, Gambino G, Schubert A (2007) A Cetyltrimethyl ammonium bromide–based method to extract low molecular weight RNA from polysaccharide–rich plant tissues. Anal Biochem 360:318–320PubMedCrossRefGoogle Scholar
  6. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine tree. Plant Mol Biol Report 11:113–116CrossRefGoogle Scholar
  7. Chomczynski P, Sacchi N (1987) Single–step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal Biochem 162:156–159PubMedCrossRefGoogle Scholar
  8. Claros MG, Canovas FM (1998) Rapid high-quality RNA preparation from Pine. Plant Mol Biol Report 16:9–18.CrossRefGoogle Scholar
  9. Gambino G, Gribaudo I (2006) Simultaneous detection of nine grapevine viruses by multiplex RT–PCR with coamplification of a plant RNA internal control. Phytopathol 96:1223–1229CrossRefGoogle Scholar
  10. Gambino G, Bondaz J, Gribaudo I (2006) Detection and elimination of viruses in callus, somatic embryos and regenerated plantlets of grapevine. Eur J Plant Pathol 114:397–404CrossRefGoogle Scholar
  11. Gambino G, Perrone I, Gribaudo I (2008) A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem Anal 19:520–525PubMedCrossRefGoogle Scholar
  12. Geuna F, Hartings H, Scienza A (1998) A new method for rapid extraction of high quality RNA from recalcitrant tissues of grapevine. Plant Mol Biol Report 16:61–67.CrossRefGoogle Scholar
  13. Iandolino AB, Goes da Silva F, Lim H, Choi H, Williams LE, Cook DR (2004) High–quality RNA, cDNA, and derived EST libraries from grapevine (Vitis vinifera L.). Plant Mol Biol Report 22:269–278CrossRefGoogle Scholar
  14. Kolosova N, Miller B, Ralph S, Ellis BE, Douglas C, Ritland K, Bohlmann J (2004) Isolation of high–quality RNA from gymnosperm and angiosperm trees. Biotech 36:821–824Google Scholar
  15. Le Provost G, Herrera R, Paiva JAP, Chaumeil P, Salin F, Plomion C (2007) A micromethod for high throughput RNA extraction in forest trees. Biol Res 40:291.297PubMedGoogle Scholar
  16. Li B, Wang B, Tang K, Liang Y, Wang J, Wei J (2006) A simple and convenient approach for isolating RNA from highly viscous plant tissue rich in polysaccharides. Colloids Surf B Biointerfaces 49:101–105PubMedCrossRefGoogle Scholar
  17. Liu JJ, Goh CJ, Loh CS, Liu P, Pua EC (1998) A method for isolation of total RNA from fruit tissues of banana. Plant Mol Biol Report 16:1–6CrossRefGoogle Scholar
  18. Loulakakis KA, Roubelakis–Angelakis KA, Kanellis AK (1996) Isolation of functional RNA from grapevine tissues poor in nucleic acid content. Am J Enol Vitic 47:181–185Google Scholar
  19. Meisel L, Fonseca B, Gonzalez S, Baeza-Yates R, Cambiazo V, Campos R, Gonzalez M, Orellana A, Retamales J, Silva H (2005) A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses. Biol Res 38:83–88PubMedCrossRefGoogle Scholar
  20. Moser C, Gatto P, Moser M, Pindo M, Velasco R (2004) Isolation of functional RNA from small amounts of different grape and apple tissues. Mol Biotechnol 26:95–100PubMedCrossRefGoogle Scholar
  21. Or E, Belausov E, Popilevsky I, Ben Tal Y (2000) Changes in endogenous ABA level in relation to the dormancy cycle in grapevine grown in hot climate. J Hortic Sci Biotechnol 75:190–194Google Scholar
  22. Ophir R, Pang X, Halaly T, Venkateswari J, Lavee S, Galbraith D, Or E (2009) Gene-expression profiling of grape bud response to two alternative dormancy-release stimuli expose possible links between impaired mitochondrial activity, hypoxia, ethylene-ABA interplay and cell enlargement. Plant Mol Biol 71:403–423PubMedCrossRefGoogle Scholar
  23. Rott ME, Jelkmann W (2001) Characterization and detection of several filamentous viruses of cherry: adaptation of an alternative cloning method (DOP–PCR), and modification of an RNA extraction protocol. Eur J Plant Pathol 107:411–420CrossRefGoogle Scholar
  24. Salzman RA, Fujita T, Zhu-Salzman K, Hasegawa PM, Bressan RA (1999) An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol Biol Report 17:11–17CrossRefGoogle Scholar
  25. Scott DL Jr, Clark CW, Deahl KL, Prakash CS (1998) Isolation of functional RNA from periderm tissue of potato tubers and sweet potato storage roots. Plant Mol Biol Report 16:3–8CrossRefGoogle Scholar
  26. Tattersall EAR, Ergul A, Alkayal F, DeLuc L, Cushman JC, Cramer GR (2005) Comparison of methods for isolating high–quality RNA from leaves of grapevine. Am J Enol Vitic 56:400–406Google Scholar
  27. Thomas P, Schiefelbein JW (2002) Improved method for purification of RNA from stem tissue of grapevine and its use in mRNA profiling. Am J Enol Vitic 53:231–234Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Atiako Kwame Acheampong
    • 1
  • Ariel Rotman
    • 1
  • Chuanlin Zheng
    • 1
  • Alexandra Keren
    • 1
  • Tamar Halaly
    • 1
  • Omer Crane
    • 1
  • Aliza Ogrodovitch
    • 1
  • Etti Or
    • 2
  1. 1.Institute of Plant Sciences, A.R.O. Volcani CenterBet DaganIsrael
  2. 2.Department of Horticulture, Agricultural Research Organisation, Department of Fruit Tree SciencesThe Volcani CenterBet DaganIsrael

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