Improvement of an RNA purification method for grapevine (Vitis vinifera L.) suitable for cDNA library construction
- 146 Downloads
A method is described, which consistently yields high quality total RNA from grapevine. Dissolving of crude RNA pellets in borate-containing buffer, instead of normally used water before a selective lithium chloride precipitation, was found to be a critical step, leading to a 2.5-fold increase of yield. The resulting RNA preparations were suitable for standard downstream applications and also for cDNA library construction. The method worked efficiently and reproducibly and could easily be scaled from milligram to gram quantities of plant material grown in hydroponic culture, sandy soil or Perlite. It was applied to different kinds of grapevine tissues (leaves, stem) and, after additional adaptation of the protocol, to roots.
KeywordscDNA library construction Grape RNA extraction Tris–borate Vitis vinifera L.
Ethylene diamine tetra-acetic acid
Elongation factor one alpha
Reverse transcription-polymerase chain reaction
Sodium dodecyl sulphate
- Boss PK, Davies C (2001) Molecular biology of sugar and anthocyanin accumulation in grape berries. In: Roubelakis-Angelakis KA (ed) Molecular biology and biotechnology of the grapevine. Kluwer Academic Publishers, Netherlands, pp 1–33Google Scholar
- Daldoul S, Höfer MU, Linhard C, Jallouli N, Mliki A, Reustle GM, Ghorbel A (2008) Expression analysis of salt stress responsive genes in grapevines. In: Abdelly C, Öztürk M, Ege Ashraf M, Grignon C (eds) Biosaline agriculture and high salinity tolerance. Birkhäuser Verlag AG, Switzerland, pp 297–303CrossRefGoogle Scholar
- Jaillon O, Aury J-M, Noel B, Policriti A, Clepet C, Casagrande A, Nathalie Choisne N, Aubourg S, Vitulo N, Jubin C, Vezzi A, Legeai F, Hugueney P, Dasilva C, Horner D, Mica E, Jublot D, Poulain J, Bruyaire C, Billault A, Segurens B, Gouyvenoux M, Ugarte E, Federica Cattonaro F, Anthouard V, Vico V, Fabbro CD, Alaux M, Di Gaspero G, Dumas V, Felice N, Paillard S, Juman I, Moroldo M, Scalabrin S, Canaguier A, Le Clainche I, Giorgio Malacrida G, Durand E, Pesole G, Laucou V, Philippe Chatelet P, Didier Merdinoglu D, Delledonne M, Pezzotti M, Lecharny A, Scarpelli C, Artiguenave F, Pe ME, Valle G, Morgante M, Caboche M, Adam-Blondon A-F, Weissenbach J, Quetier F, Wincker P (2007) The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature 449:463–467. doi:10.1038/nature06148 PubMedCrossRefGoogle Scholar
- Renault AS, Deloire A, Letinois I, Kraeva E, Tesniere C, Ageorges A, Redon C, Bierne J (2000) Beta-1,3-glucanase gene expression in grapevine leaves as a response to infection with Botrytis cinerea. Am J Enol Vitic 5:81–87Google Scholar
- 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(4):400–406Google Scholar
- 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
- Troggio M, Pezzulli S, Pindo M, Malacarne G, Fontana P, Moreira FM, Costantini L, Grando MS, Viola R, Velasco R (2008) Beyond the genome, opportunities for a modern viticulture: a research overview. Am J Enol Vitic 59(2):117–127Google Scholar