Intercalibration of four spectrofluorometric protocols for measuring RNA/DNA ratios in larval and juvenile fish

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 mDNA/mRNA). 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 mDNA/mRNA 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