Abstract
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.
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Claims 10
Claim · model-written summary
The four buffers differed in performance.
Verbatim
e0“Clear differences among buffers were observed.”
e1“seven plant species”
Claim · model-written summary
Except for O. pes-caprae, any buffer gave acceptable results for all species.
- Taxon
- O. pes-caprae
Oxalis pes-capraee0 - Outcome
- acceptable resultse0
- Comparator
- any buffere0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“With the exception of O. pes-caprae, any buffer provided acceptable results for all species.”
Claim · model-written summary
LB01 generally ranked among the best buffers.
Verbatim
e0“LB01 and Otto's were generally the best buffers”
e1“seven plant species”
Claim · model-written summary
Otto’s generally ranked among the best buffers and performed better with low-DNA-content species.
Verbatim
e0“LB01 and Otto's were generally the best buffers, with Otto's buffer providing better results in species with low DNA content.”
e1“seven plant species”
Claim · model-written summary
Galbraith’s buffer produced satisfactory results.
Verbatim
e0“Galbraith's buffer led to satisfactory results”
e1“seven plant species”
Claim · model-written summary
Tris.MgCl2 was generally the worst buffer.
Verbatim
e0“Tris.MgCl2 was generally the worst”
e1“seven plant species”
Claim · model-written summary
Tris.MgCl2 gave the best histograms for C. australis.
- Taxon
- C. australis
Celtis australise0 - Outcome
- best histogramse0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“it yielded the best histograms in C. australis”
Claim · model-written summary
Combined forward and side scatter analysis produced a fingerprint for each buffer.
Verbatim
e0“A combined analysis of FS and SS provided a 'fingerprint' for each buffer.”
e1“seven plant species”
Claim · model-written summary
Variation across days exceeded variation across operators.
Verbatim
e0“The variation between days was more significant than the variation between operators.”
e1“seven plant species”
Claim · model-written summary
No buffer performed best across every species.
Verbatim
e0“none of the buffers worked best with all species”
e1“seven plant species”