Methodology

Procedures

One step procedure using Lysis bufer LB01, Tris-MgCl₂ buffer, and Seed buffer

• Add fluorochrome (DAPI, 4 µg/ml or propidium iodide + RNase, both 50 µg/ml) to the selected buffer; ß-mercaptoethanol (2 µl/ml) might be used to avoid polyphenolics oxidation.
  

  (DAPI-staining: 25 ml buffer + 1 ml DAPI stock solution + 50 µl ß-mercaptoethanol;
  Propidium iodide-staining: 20 ml buffer + 1 ml PI stock solution + 1 ml RNase stock solution + 40 µl ß-mercaptoethanol)

• Chop young intact leaf tissues (typically 1 cm²) or seeds with a new razor blade in a petri dish containing 1 ml of ice-cold buffer.
• Filter the suspension through a 42 µm nylon mesh.
• Incubate in the dark at 4°C (a few minutes to one hour) with occasional shaking (incubation period that gives the lowest background and CV should be selected).
• Analyse relative DNA content of isolated nuclei.

Two-step procedure using Otto I+II buffers

• Add fluorochrome (DAPI, 4 µg/ml or propidium iodide + RNase, both 50 µg/ml) to the Otto II buffer; ß-mercaptoethanol (2 µl/ml) might be used to avoid polyphenolics oxidation.
  

  (DAPI-staining: 25 ml Otto II buffer + 1 ml DAPI stock solution + 50 µl ß-mercaptoethanol;
  Propidium iodide-staining: 20 ml Otto II buffer + 1 ml PI stock solution + 1 ml RNase stock solution + 40 µl ß-mercaptoethanol)

• Chop young intact leaf tissues (typically 1 cm²) with a new razor blade in a petri dish containing 1 ml of ice-cold Otto I buffer
.
• Filter the suspension through a 42 µm nylon mesh
.
• Centrifuge the sample (150 g / 5 min).
• Remove the supernatant (leave ca 100 µl of the liquid), resuspend the nuclei by gentle shaking, add 100 µl of fresh ice-cold Otto I buffer, shake gently.
• Incubate at room temperature for 5 - 120 min with occasional shaking (incubation period that gives the lowest background and CV should be selected). If necessary, samples may be stored for prolonged period (several hours).
• Add 1 ml of Otto II buffer supplemented with fluorochrome, shake well.
• Store in the dark at room temperature (generally 5 - 15 min).
• Analyse relative DNA content of isolated nuclei.
  

  Simplified procedure (without centrifugation) may be used. In this modification, use only 0.5 ml of Otto I buffer (step 1) and avoid steps 4 and 5.

General recommendations for nuclear DNA content estimation:

• Internal standardization should always be used.
• Nuclear DNA contents of the internal standard and the sample should be reasonably close (but not overlapping or extremely close).
• The same internal standard is recommended for all taxa belonging to the group examined. If more internal standards are necessary (to span the range of DNA amounts), the secondary standard must be calibrated against the primary one (it is not a good idea to use tabulated values).
• Young (but not premature), intact, parasite- and disease-free leaves of both sample and internal standard should be used. The plants should be cultivated under the same conditions to eliminate potential negative effect of different levels of secondary metabolites.
• Intercalating propidium iodide (+ RNAse) at concentration between 50 - 70 µg / ml (pH 7.2 - 7.4) must be chosen as a fluorochrome for DNA content determination in absolute units (more sensitive DAPI with A-T preference provides only expression in relative values).
• Each measurement must be repeated at least three times on different days (repetitions on the same day are useless). If the variation between individual runs exceeds some critical value (e.g. two percent, potentially more in species with low DNA contents), additional measurements should be performed. The most out-layered measurement can be avoided.
• The peaks of both the internal standard and the sample should be symmetrical (non-skewed) and of approximately the same height. The fluorescence of 5 000 - 7 000 particles should be recorded.
• The analyses must not be distorted by the presence of secondary metabolites - the mean peak position of the internal reference standard should be located on (almost) identical fluorescence channel in all measurements, either with or without the sample
• The coefficient of variation of G0/G1 peaks should be reasonably low (generally below 3 %, although higher values can be regarded as acceptable e.g. in plants possessing very small nuclear DNA content).
• All measurements must be performed on the same flow cytometer with the same light source of excitation, as small but systematic differences exist between different types of equipment the same operator is also recommended. The cytometer should be adjusted to maximum signal amplitude, minimal CV and minimal background. In Partec intruments, the gain (signal amplification) should not exceed the value of ca. 600 (ideally less than 500).
• It is advisable to measure at least three individuals per taxon to inspect potential intraspecific DNA amount variability. In wide-range studies, this point can be substituted by a realization of simultaneous analysis of several (usually 3-5) individuals. If one narrow and symmetrical peak is achieved, one representative plant can be selected and further investigated.
• Exact chromosome number should be determined for the material analysed. This point has become absolutely necessary when intraspecific variation is suspected (aneuploidy or a presence of accessory chromosomes may be responsible for that kind of variation).
• Confirmation of true variability in nuclear DNA content requires additional pieces of inspection. Simultaneous analyses of samples supposed to differ in nuclear DNA content should be performed (bifurcated peak corroborates the theory of non-identical DNA amount). An employment of two reference standards (so that the DNA content of the sample is located in-between) is another advocated approach. The peak ratio of both reference standards should be (almost) the same in all analyses, either with or without the sample.
• Each estimation should be supplemented by a herbarium voucher kept in public herbarium.
• Last but not least recommendation: the operator should stay cheerful all along the experimentation :-)

Buffers and stock solutions

Otto Buffer I (Otto 1990)

adjust volume to 200 ml
filter through a 0.22 µm filter; store at 4°C

Otto Buffer II

adjust volume to 200 ml
filter through a 0.22 µm filter, store at room temperature
re-filter before each use

Lysis buffer LB01 (Doležel et al. 1989)

adjust volume to 200 ml, adjust pH to 7.5 (1N HCl)
filter through a 0.22 µm filter, store at -20°C in 10 ml aliquots

Tris-MgCl₂ buffer (Pfosser et al. 1995)

adjust volume to 200 ml, adjust pH to 7.5 (1N HCl)
filter through a 0.22 µm filter; store at 4°C

Seed buffer (Matzk et al. 2001, modified)

adjust volume to 100 ml, adjust pH to 7.5 (1N HCl)
filter through a 0.22 µm filter; store at 4°C

DAPI stock solution (0.1 mg/ml)

dissolve in 100 ml H₂O
filter through a 0.22 µm filter, store at -20°C in 1 ml aliquots

Propidium iodide stock solution (1 mg/ml)

dissolve in 100 ml H₂O
filter through a 0.22 µm filter, store at -20°C in 1 ml aliquots

RNase stock solution (1 mg/ml)

disolve in 100 ml H₂O
filter through a 0.22 µm filter
heat to 90°C for 15 min to inactivate DNases
store at -20°C in 1 ml aliquots

References:

Plant DNA standards