AOBPreview originally published online on July 4, 2006
Annals of Botany 2006 98(3):679-689; doi:10.1093/aob/mcl141
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TECHNICAL ARTICLE |
Comparison of Four Nuclear Isolation Buffers for Plant DNA Flow Cytometry
EL21 Laboratory of Biotechnology and Cytomics, Department of Biology, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal and 2 Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovská, Olomouc, CZ-77200, Czech Republic
* For correspondence. E-mail jloureiro{at}bio.ua.pt
Received: 15 March 2006 Returned for revision: 24 April 2006 Accepted: 22 May 2006 Published electronically: 4 July 2006
• Background and Aims DNA flow cytometry requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. Various buffer formulas were developed to preserve nuclear integrity, protect DNA from degradation and facilitate its stoichiometric staining. Although nuclear isolation buffers differ considerably in chemical composition, no systematic comparison of their performance has been made until now. This knowledge is required to select the appropriate buffer for a given species and tissue.
• Methods Four common lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2) were used to prepare samples from leaf tissues of seven plant species (Sedum burrito, Oxalis pes-caprae, Lycopersicon esculentum, Celtis australis, Pisum sativum, Festuca rothmaleri and Vicia faba). The species were selected to cover a wide range of genome sizes (1·30–26·90 pg per 2C DNA) and a variety of leaf tissue types. The following parameters were assessed: forward (FS) and side (SS) light scatters, fluorescence of propidium iodide-stained nuclei, coefficient of variation of DNA peaks, presence of debris background and the number of nuclei released from sample tissue. The experiments were performed independently by two operators and repeated on three different days.
• Key Results Clear differences among buffers were observed. With the exception of O. pes-caprae, any buffer provided acceptable results for all species. LB01 and Otto's were generally the best buffers, with Otto's buffer providing better results in species with low DNA content. Galbraith's buffer led to satisfactory results and Tris.MgCl2 was generally the worst, although it yielded the best histograms in C. australis. A combined analysis of FS and SS provided a ‘fingerprint’ for each buffer. The variation between days was more significant than the variation between operators.
• Conclusions Each lysis buffer tested responded to a specific problem differently and none of the buffers worked best with all species. These results expand our knowledge on nuclear isolation buffers and will facilitate selection of the most appropriate buffer depending on species, tissue type and the presence of cytosolic compounds interfering with DNA staining.
Key words: Angiosperms, flow cytometry, genome size, lysis buffers, nuclear DNA content, nuclear isolation buffers, propidium iodide, stoichiometric error
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