Plant genome size estimation by flow cytometry:: Inter-laboratory comparison
Plant genome size estimation by flow cytometry:: Inter-laboratory comparison
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DOI：
10.1006/anbo.1998.0730
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发表时间：
1998-12-01
期刊：
影响因子：
4.2
通讯作者：
Obermayer, R
中科院分区：
文献类型：
--
作者：
Dolezel, J;Greilhuber, J;Obermayer, R
流式细胞术是一种方便、快速的方法，已广泛用于估计植物核基因组大小。与一般预期相反，不同实验室获得的结果显示出一些惊人的差异。该联合实验的目的是测试方法的可靠性和再现性。小心避免由于细胞核中 DNA 的数量、细胞核分离程序或仪器类型造成的偏差。对九种植物的核 DNA 含量进行了估计，这些植物代表了典型的基因组大小范围（2C = 约 0.3-30 pg DNA）。参与本研究的四个实验室均使用不同的缓冲液和/或程序进行细胞核分离。两个实验室使用的是基于灯的仪器，而另外两个实验室则使用基于激光的仪器。用碘化丙啶（DNA 嵌入剂）进行细胞核染色后获得的结果与使用 Feulgen 光密度测定法获得的结果非常吻合。另一方面，用 DAPI 染色后获得的结果（优先结合 AT 丰富的区域）与使用 Feulgen 光密度测定法获得的结果不一致。使用各个仪器获得的数据之间存在微小但具有统计学意义的差异。同一类型仪器之间的差异可以忽略不计，而基于灯和激光的仪器之间观察到更大的差异。激光仪器获得的荧光强度比高于基于灯的细胞仪或福尔根光密度测定法获得的荧光强度比。本研究获得的结果表明，使用 DNA 嵌入剂的细胞计数法是估计植物核基因组大小的可靠方法。然而，该研究证实迫切需要就标准达成一致。鉴于不同类型的流式细胞仪之间存在微小但系统性的差异，因此应在同一实验室并使用同一仪器对基因组大小的微小差异进行分析。 (C) 1998 年植物学公司年鉴。
Flow cytometry is a convenient and rapid method that has been used extensively for estimation of nuclear genome size in plants. In contrast to general expectations, results obtained in different laboratories showed some striking discrepancies. The aim of this joint experiment was to test the reliability and reproducibility of methods. Care was taken to avoid a bias due to the quantity of DNA in the nucleus, the procedure for nuclei isolation or the type of instrument. Nuclear DNA content was estimated in nine plant species representing a typical range of genome size (2C = approx. 0.3-30 pg DNA). Each of the four laboratories involved in this study used a different buffer and/or procedure for nuclei isolation. Two laboratories used are lamp-based instruments while the other two used laser-based instruments. The results obtained after nuclei staining with propidium iodide (a DNA intercalator) agreed well with those obtained using Feulgen densitometry. On the other hand, results obtained after staining with DAPI (binding preferentially to AT-rich regions) did not agree with those obtained using Feulgen densitometry. Small, but statistically significant, differences were found between data obtained with individual instruments. Differences between the same type of instruments were negligible, while larger differences were observed between lamp- and laser-based instruments. Ratios of fluorescence intensity obtained by laser instruments were higher than those obtained by lamp-based cytometers or by Feulgen densitometry. The results obtained in this study demonstrate that how cytometry with DNA intercalators is a reliable method for estimation of nuclear genome size in plants. However, the study confirmed an urgent need for an agreement on standards. Given the small but systematic differences between different types of flow cytometers, analysis of very small differences in genome size should be made in the same laboratory and using the same instrument. (C) 1998 Annals of Botany Company.