Plant Molecular Biology Reporter

, Volume 18, Issue 4, pp 369–376

An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides

  • H. H. Gehrig
  • K. Winter
  • J. Cushman
  • A. Borland
  • T. Taybi
Protocols

Abstract

Isolation of high quality, high molecular weight RNA from plant tissues is complicated by high levels of polyphenols and polysaccharides which bind to and/or co-precipitate with RNA. Using high molecular weight polyethylene glycol (HMW-PEG), RNA was successfully isolated from plant species in which other RNA extraction methods and commercially available kits failed to deliver suitable results. We tested various buffer systems and isolation conditions with and without PEG or PVP (polyvinylpyrrolidone) using tissue from species ofAloe, Ananas, Clusia, Euphorbia, Kalanchoe, Opuntia, andPyrrosia, all of which contain high amounts of phenolic compounds and/or polysaccharides. HMW-PEG was found to be more effective than PVP in removing these compounds. RNA extraction using HMW-PEG resulted in RNA of high quality from all species investigated, as indicated by UV light absorption profiles, and also yielded PCR amplification products after reverse transcription.

Key words

Aloe Ananas Clusia crassulacean acid metabolism Euphorbia Kalanchoe Opuntia phenolic compounds Pyrrosia RNA extraction 

Abbreviations

CAM

crassulacean acid metabolism

DEPC

diethylpyrocarbonate

EtBr

ethidium bromide

FW

fresh weight

GHCL

guanidinium hydrochloride

GITC

guanidinium isothiocyanate

HMW-PEG

high-molecular-weight polyethylene glycol

NaAc

sodium-acetate

PCI

phenol/chloroform/isoamyl-alcohol-mix

PVP

polyvinylpyrrolidone

RT

room temperature

RT-PCR

reverse-transcription-polymerase chain reaction

TRIR

Trizol-reagent

UV

ultraviolet

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Cathala G, Savouret JF, Mendez B, West BL, Karin M, Martial JA and Baxter JD (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2: 329–335.PubMedCrossRefGoogle Scholar
  2. Chang S, Puryear J and Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Reptr 11: 113–116.CrossRefGoogle Scholar
  3. Chirgwin JM, Pryzbyla AE, MacDonald RJ and Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem 18: 5294–5299.CrossRefGoogle Scholar
  4. Chomczynski P and Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159.PubMedCrossRefGoogle Scholar
  5. Glisin V, Crkvenjakov R and Byus D (1974) Ribonucleic acid isolation by caesium chloride centrifugation. Biochem 13: 2633–2637.CrossRefGoogle Scholar
  6. Gruffat D (1998) Isolation of RNA from mammalian cells: application to large mRNA. In: Siebert PD and Larrick JW (eds), Gene cloning and analysis by RT-PCR. BioTechniques 23: 35–55.Google Scholar
  7. Han JH, Stratowa C and Rutter WJ (1987) Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning. Biochem 26: 1617–1625.CrossRefGoogle Scholar
  8. Loomis WD (1974) Overcoming problems of phenolic and quinones in the isolation of plant enzymes and organelles. Meth Enzymol 31: 528–545.PubMedCrossRefGoogle Scholar
  9. Salzman RA, Fujita T, Zhu-Salzman K, Hasegawa PM and Bressan RA (1999) An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates. Plant Mol Biol Reptr 17: 11–17.CrossRefGoogle Scholar
  10. Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, New York, U.S.A.Google Scholar
  11. Tesnier C and Vayda ME (1991) Method for isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates. Plant Mol Biol Reptr 9: 242–251.CrossRefGoogle Scholar
  12. Woodhead M, Taylor MA, Davies HV, Brennan RM and McNicol RJ (1997) Isolation of RNA from blackcurrant (Ribes nigrum L.) fruit. Mol Biotechnol 7(1): 1–4.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2000

Authors and Affiliations

  • H. H. Gehrig
    • 1
  • K. Winter
    • 1
  • J. Cushman
    • 2
  • A. Borland
    • 3
  • T. Taybi
    • 3
  1. 1.Smithsonian Tropical Research InstituteBalboaRepublic of Panama
  2. 2.University of Nevada, MS200RenoUSA
  3. 3.Dept. of Agricultural and Environmental SciencesUniversity of NewcastleNewcastle upon TyneU.K.

Personalised recommendations