Intercalibration of four spectrofluorometric protocols for measuring RNA/DNA ratios in larval and juvenile fish
Intercalibration of four spectrofluorometric protocols for measuring RNA/DNA ratios in larval and juvenile fish
复制标题
DOI：
10.4319/lom.2006.4.153
复制
发表时间：
2006-05-01
影响因子：
2.7
通讯作者：
Suthers, Iain M.
中科院分区：
文献类型：
--
作者：
Caldarone, Elaine M.;Clemmesen, Catriona M.;Suthers, Iain M.
组织RNA / DNA比值（R/D）是一种被广泛应用于幼鱼近期生长和营养状况的指标。然而，到目前为止，还没有采用测量核酸的标准技术。由于方法上的细节可能会影响R/D的估计，使用不同分析方案的研究人员无法直接比较比率。在这里，我们报告了一项国际实验室间校准的4个荧光光谱测定方案的结果，以定量核酸。5名研究人员每人提供5份组织样品和2份标准品（通用标准品）的重复组，用各自的方法和标准进行分析。评估了两种方法来减轻观察到的值差异：1)使用共同的核酸标准，2)标准化到一个共同的斜率比(DNA标准曲线的斜率/ RNA标准曲线的斜率或m（DNA)/m(RNA)）。采用共同标准略微减少了协议之间的可变性，但并没有克服这个问题。当组织R/ d基于普通的m(DNA)/m（RNA）斜率比进行标准化时，归因于分析方案的方差从57.1%急剧下降到3.4%。我们建议提供标准曲线斜率的比值，以促进使用不同荧光光谱法分析鱼类核酸的实验室之间的研究/开发结果的可比性。
The ratio of tissue RNA to DNA (R/D) is a widely used index of recent growth and nutritional condition in larval and juvenile fish. To date, however, no standard technique for measuring nucleic acids has been adopted. Because methodological details can affect the estimate of R/D, researchers using different analytical protocols have been unable to compare ratios directly. Here, we report on the results of an international interlaboratory calibration of 4 spectrofluorometric protocols to quantify nucleic acids. Replicate sets of 5 tissue samples and 2 standards ( common standards) were supplied to each of 5 researchers for analysis with their own methods and standards. Two approaches were evaluated for mitigating the observed differences in values: 1) the use of common nucleic acid standards and 2) standardizing to a common slope ratio ( slope of DNA standard curve/slope of RNA standard curve or m(DNA)/m(RNA)). Adopting common standards slightly reduced the variability among protocols but did not overcome the problem. When tissue R/Ds were standardized based on a common m(DNA)/m(RNA) slope ratio, the variance attributed to analytical protocol decreased dramatically from 57.1% to 3.4%. We recommend that the ratio of the slopes of the standard curves be provided to facilitate intercomparability of R/D results among laboratories using different spectrofluorometric methods for the analysis of nucleic acids in fish.