Doležel et al. 1998

Doležel J, Greilhuber J, Lucretti S, Meister A, Lysák MA, Nardi L, Obermayer R. 1998. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Annals of Botany 82 (Supplement A): 17-26.

These authors compared pairwise ratios of measured genome sizes of nine species of plants covering a genome size range of 0.3-30pg, between four laboratories in central Europe (Austria, Czech Republic, Germany, Italy), using flow cytometry. The purpose of this “exercise” was to test the reliability and variability of flow cytometry for genome-size measurement in plants. Each lab used its own method of nuclei isolation and staining; one lab also measured these plant species using Feulgen densitometry. Two labs used laser-based flow cytometers, two used mercury arc-lamp-based flow cytometers. All labs used propidium iodide (PI), a DNA-intercalating agent that is not biased by sequence; one of each type of machine was also used with DAPI, which preferentially binds to AT-rich regions of chromosomes.

Their primary findings were 1. intra-lab variation was very low, 2. inter-lab variation was low but statistically significant (partly because intra-lab variation was so low), and 3. a consistent difference was found between mercury arc-lamp and laser-based flow cytometers. Additionally, they found that DAPI was not suitable for genome size estimation because of differences between plant species in GC contents. Variation in nuclei-isolating buffers did not have measureable effects on the results.

They make two key recommendations for future research into plant genome size variation using flow cytometry:
1. All samples should be analysed in one lab using one instrument. Replication of one lab’s findings by another should analyse whole sets of samples for reliable ratio comparisons.
2. Inter-lab and machine-type differences should be considered when evaluating published reports of genome size variation in plants.
Both of these recommendations underscore the need for detailed, clear Methods & Materials sections in scientific publications, including the need to identify staining protocols, instrument types, and similar fine details.

Finally, they urge a broad agreement on reference standards (i.e. species used as standards during measurement) and their calibration, to allow higher precision in estimated genome size variation among and within plant species.