Improvement of an RNA purification method for grapevine (Vitis vinifera L.) suitable for cDNA library construction
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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
The authors thank Claudia Linhard (AlPlanta) for the excellent technical assistance and Manfred Jutzi (DLR Rheinpfalz, Neustadt a.d. Weinstrasse, Germany) for performing the statistical analyses. This work was supported in part by an ICGEB research grant (CRP/TUN06-01). S. Daldoul was also supported by a research grant of the German Academic Exchange Service (DAAD).
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