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An improved method for the rapid isolation of RNA from Arabidopsis and seeds of other species high in polyphenols and polysaccharides

Published online by Cambridge University Press:  07 August 2018

Steven Footitt ,
Sajjad Awan  and
William E. Finch-Savage
Steven Footitt*
Affiliation:
School of Life Sciences, Wellesbourne Campus, Warwick University, Wellesbourne, Warwick CV35 9EF, UK
Sajjad Awan
Affiliation:
School of Life Sciences, Wellesbourne Campus, Warwick University, Wellesbourne, Warwick CV35 9EF, UK
William E. Finch-Savage
Affiliation:
School of Life Sciences, Wellesbourne Campus, Warwick University, Wellesbourne, Warwick CV35 9EF, UK
*
Author for correspondence: Steven Footitt, Email: sfootitt@outlook.com
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Abstract

Seeds are notoriously high in polyphenols and polysaccharides, which reduce RNA quality and yield, and interfere with downstream applications. We present simple modifications to a rapid RNA extraction protocol for use with seeds. The inclusion of polyethylene glycol in place of polyvinylpyrrolidone reduced polyphenol and polysaccharide contamination. In addition, replacing NaCl with KCl improved the RNA yield from Arabidopsis seeds still bound by mucilage. On extraction of Arabidopsis seed recovered from field soils clean RNA pellets with no accompanying gelatinous matrix (polysaccharide) were seen, with A260/230 ratios greater than 1.8 confirming the lack of polysaccharide carry-over. When Brassica oleracea and Sinapis arvensis seeds were extracted, 260/230 ratios greater than 1.8 were seen. RNA yields in excess of 10 µg per 100 mg seed suitable for RT-QPCR were obtained.

Keywords

Arabidopsis thalianaBrassicapolysaccharideRNA extractionseeds
Type
Technical Update
Information
Seed Science Research , Volume 28 , Issue 4 , December 2018 , pp. 360 - 364
Copyright
Copyright © Cambridge University Press 2018 

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References

Asif, MH, Dhawan, P and Nath, P (2000) A simple procedure for the isolation of high quality RNA from ripening banana fruit. Plant Molecular Biology Reporter 18, 109–115.Google Scholar
Awan, S, Footitt, S and Finch-Savage, WE (2018) Interaction of maternal environment and allelic differences in seed vigour genes determines seed performance in Brassica oleracea. The Plant Journal 94, 1098–1108.Google Scholar
Bera, BC, Foster, AB and Stacey, M (1955) Observations on the properties of cetyltrimethylammonium salts of some acidic polysaccharides. Journal of the Chemical Society (Resumed) 3788–3793.Google Scholar
Birtic, S and Kranner, I (2006) Isolation of high-quality RNA from polyphenol-,polysaccharide- and lipid-rich seeds. Phytochemical Analysis 17, 144–148.Google Scholar
Cadman, CS, Toorop, PE, Hilhorst, HW and Finch-Savage, WE (2006) Gene expression profiles of Arabidopsis Cvi seeds during dormancy cycling indicate a common underlying dormancy control mechanism. Plant Journal 46, 805–822.Google Scholar
Carrera, E, Holman, T, Medhurst, A, Dietrich, D, Footitt, S, Theodoulou, FL and Holdsworth, MJ (2008) Seed after-ripening is a discrete developmental pathway associated with specific gene networks in Arabidopsis. Plant Journal 53, 214–224.Google Scholar
Chang, S, Puryear, J and Cairney, J (1993) A simple and effcient method for isolating RNA from pine trees. Plant Molecular Biology Reporter 11, 113–116.Google Scholar
Fang, G, Hammar, S and Grumet, R (1992) A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biotechniques 13, 52–54.Google Scholar
Footitt, S, Douterelo-Soler, I, Clay, H and Finch-Savage, WE (2011) Dormancy cycling in Arabidopsis seeds is controlled by seasonally distinct hormone-signaling pathways. Proceedings of the National Academy of Sciences of the USA 108, 20236–20241.Google Scholar
Footitt, S, Huang, Z, Clay, HA, Mead, A and Finch-Savage, WE (2013) Temperature, light and nitrate sensing coordinate Arabidopsis seed dormancy cycling, resulting in winter and summer annual phenotypes. Plant Journal 74, 1003–1015.Google Scholar
Footitt, S, Müller, K, Kermode, AR and Finch-Savage, WE (2015) Seed dormancy cycling in Arabidopsis: chromatin remodelling and regulation of DOG1 in response to seasonal environmental signals. Plant Journal 81, 413–425.Google Scholar
Gehrig, HH, Winter, K, Cushman, J, Borland, A and Taybi, T (2000) An improved RNA isolation method for succulent plant species rich in polyphenols and polysaccharides. Plant Molecular Biology Reporter 18, 369–376.Google Scholar
Murray, MG and Thompson, W (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8, 4321–4326.Google Scholar
Sharma, AD, Gill, PK and Singh, P (2003) RNA isolation from plant tissues rich in polysaccharides. Analytical Biochemistry 314, 319–321.Google Scholar
Smidsrød, O and Haug, A (1967) Precipitation of acidic polysaccharides by salts in ethanol–water mixtures. In Journal of Polymer Science, Polymer Symposia 16, 1587–1598. Wiley Subscription Services, Inc., a Wiley Company.Google Scholar
Smidsrød, O, Larsen, B, Pernas, AJ and Haug, A (1967) The effect of alkali treatment on the chemical heterogeneity and physical properties of some carrageenans. Acta Chemica Scandinavica 21, 2585–2598.Google Scholar
Vicient, CM and Delseny, M (1999) Isolation of total RNA from Arabidopsis thaliana seeds. Analytical Biochemistry 268, 412–413.Google Scholar
Xu, J, Aileni, M, Abbagani, S and Zhang, P (2010) A reliable and efficient method for total RNA isolation from various members of spurge family (Euphorbiaceae). Phytochemical Analysis 21, 395–398.Google Scholar
Wan, CY and Wilkins, TA (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Analytical Biochemistry 223, 7–12.Google Scholar