Abstract
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.
Abbreviations
- DEPC:
-
Diethyl pyrocarbonate
- EDTA:
-
Ethylene diamine tetra-acetic acid
- EF1α:
-
Elongation factor one alpha
- FW:
-
Fresh weight
- PVPP:
-
Polyvinylpolypyrrolidone
- P:C:I:
-
Phenol:chloroform:isoamylalcohol
- RT-PCR:
-
Reverse transcription-polymerase chain reaction
- SD:
-
Standard deviation
- SDS:
-
Sodium dodecyl sulphate
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Acknowledgments
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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Communicated by H. Janska.
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Daldoul, S., Chenenanoui, S., Mliki, A. et al. Improvement of an RNA purification method for grapevine (Vitis vinifera L.) suitable for cDNA library construction. Acta Physiol Plant 31, 871–875 (2009). https://doi.org/10.1007/s11738-009-0309-0
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DOI: https://doi.org/10.1007/s11738-009-0309-0