Abstract
Commonly used methods for extraction of RNA from plants are not effective for isolation of high quality RNA from the pigmented seed coats of soybeans that produce procyanidins (tannins) during seed coat development. We demonstrate a significant modification of the phenol-LiCl method that yields high quality RNA from a black seed coat variety. In this method, seed coat material was ground in a buffer containing a high concentration of bovine serum albumin (100 mg BSA/50 mg of lyophilized seed coats) to competitively inhibit proanthocyanidin binding. The presence of hydrated insoluble polyvinylpoly-pyrrolidone (PVPP) was also necessary to bind proanthocyanidins and remove them from solution. Proteinase K was added to digest the remaining BSA, and phenol extraction was used to remove both the proteins and small molecular weight complexes formed by BSA and proanthocyanidins. After LiCl and ethanol precipitations, the RNA quality was examined by UV absorbance spectra, gel electrophoresis, and hybridization. Using this method, good quality RNA can be extracted from pigmented seed coats of soybean varieties that are homozygous for the recessivei allele and also contain the dominantT gene that results in production of procyanidins in the seed coat. The method is also effective for tissues from other plant species that contain abundant polyphenolic compounds.
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Abbreviations
- PVPP:
-
polyvinylpolypyrrolidone
References
Anderson, R.A. and J.A. Sowers. 1968. Optimum conditions for binding of plant phenols to insoluble polyvinylpyrrolidone. Phytochemistry 7:293–301.
Buzzell, R.I., B.R. Buttery, and D.C. MacTavish. 1987. Biochemical genetics of black pigmentation of soybean seed. J. Hered. 78:53–54.
Chomczynski, P. and N. Sacchi, 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159.
Feinberg, A.P. and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction fragments to high specific activity. Anal. Biochem. 132:6–13.
Gaudette, M.F. and Crain W.R. 1991. A simple method for quantifying specific mRNAs in small numbers of early mouse embryos. Nucl. Acids. Res. 19:1879–1884.
Hagerman, A.E. and L.G. Butler. 1981. The specificity of proanthocyanidin-protein interactions. J. Biol. Chem. 256:4494–4497.
King, E.E. 1971. Extraction of cotton leaf enzymes with borate. Phytochem. 10:2337–2341.
Lindstrom, J.T. and L.O. Vodkin. 1991. A soybean cell wall protein is affected by seed color genotype. Plant Cell 3:561–571.
Lizardi, P.M. (1983) Methods for the preparation of messenger RNA. Method. Enz. 96:24–38.
Lizardi, P.M. and A. Engelberg. 1979. Rapid isolation of RNA using proteinase K and sodium perchlorate. Anal. Biochem. 98:116–122.
Logemann, J., J. Schell, and L. Willmitzer. 1987. Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163:16–20.
Loomis, W.D. 1968. Removal of phenolic compounds during the isolation of plant enzymes. Method. Enz. 13:555–563.
Loomis, W.D. 1974. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Method. Enz. 31:528–544.
McCarty, D.R. 1986. A simple method for extraction of DNA from maize tissue. Maize Genet. Coop. Newsl. 60:61.
Newbury, H.J. and J.V. Possingham. 1977. Factors affecting the extraction of intact ribonucleic acid from plant tissues containing interferring phenolic compounds. Plant Physiol. 60:543–547.
Nicholas, C.D., J.T. Lindstrom, and L.O. Vodkin. 1993. Quantitative and qualitative variation of a cell wall protein in soybean lines with anthocyanin mutations. Plant Mol. Biol. 21:145–156.
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989.Molecular Cloning: A Laboratory Manual, New York, Cold Spring Harbor Laboratory Press.
Stafford, H.A. 1990.Flavonoid Metabolism. CRC Press. Boca Raton, FL.
Rhoads, R.E., G.S. McKnight, and R.T. Schimke. 1971. Synthesis of ovalbumin in rabbit reticulocyte cell-free system programmed with hen oviduct ribonucleic acid. J. Biol. Chem. 246:7407–7410.
Tesniere, C. and M.E. Vayda. 1991. Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates. Plant Mol. Biol. Rep. 9:242–251.
Todd, J.J. and L.O. Vodkin. 1993. Pigmented soybean seed coats accumulate proanthocyanidins during development. Plant Physiol. 102:663–670.
Wang, C.-S., J.J. Todd, and L.O. Vodkin. 1994. Chalcone synthase mRNA and activity are reduced in yellow soybean seed coats with dominantI alleles, Plant Physiol, in press.
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Wang, CS., Vodkin, L.O. Extraction of RNA from tissues containing high levels of procyanidins that bind RNA. Plant Mol Biol Rep 12, 132–145 (1994). https://doi.org/10.1007/BF02668374
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DOI: https://doi.org/10.1007/BF02668374