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Fishery Bulletin 93(1). 1995 



nificant in either the CFA or FIA techniques using 

 the sarcosine extraction procedure with larval fish. 

 Recoveries of crude homogenate "spikes" added to 

 nucleic acid standards by FIA (Caldarone and 

 Buckley, 1991) and CFA (this study) are similar to 

 those reported for more purified extracts 

 (Clemmesen, 1993). McGurk and Kusser (1992) re- 

 ported higher RNA contents and RNA/DNA ratios 

 for yolk-sac herring larvae analyzed with the 

 Clemmesen method (1988) and suggested that 

 fluoresecence absorbance by yolk components may 

 be reduced by the purification steps in that assay. 

 However, when a homogenate of winter flounder yolk- 

 sac larvae was "spiked" with nucleic acid standards 

 and subjected to FIA with a sarcosine extraction pro- 

 cedure, recoveries of calf thymus DNA standards 

 remained unchanged and those for calf liver RNA 

 standards only declined by 3 to 5% (Caldarone, 

 Unpubl. data). 



RNA/DNA indices have proven useful as indica- 

 tors of condition in a wide variety of fish species. 

 When coupled with data on water temperature, and 

 calibrated with laboratory-reared larvae, estimates 

 of recent growth in the field can be obtained (Buckley, 

 1984). However, this study and others illustrate a 

 common problem with the application of fluorescent 

 techniques to estimation of nucleic acid levels in fish 

 and other biosamples, and the need for inter- 

 calibration. Given the disparity in estimates of RNA 

 and DNA contents due to the method of analysis 

 (McGurk and Kusser, 1992; this study) and choice of 

 nucleic acid standards (Caldarone and Buckley, 

 1991), no direct intermethodological comparisons of 

 data can be made without intercalibration between 

 analytical methods, as done by McGurk and Kusser 

 (1992), Clemmesen (1993), Mathers et al. (1994), and 

 this study. It is inappropriate to compare RNA/DNA 

 ratio values with published data unless the same 

 methods and standards are used. Also, the general- 

 ized growth equation in Buckley (1984) uses RNA/ 

 DNA ratios determined with an ultraviolet light ab- 

 sorption method that cannot be directly applied to 

 ratios determined with other analytical procedures 

 without running an intercalibration between the two 

 methods. Alternatively, the relation between RNA/ 

 DNA, temperature, and growth must be determined 

 for laboratory-reared larvae by using the analytical 

 method of choice before a growth equation can be 

 applied to fish larvae collected in the field. 



A general assay protocol for FIA and CFA is pre- 

 sented in Figure 3. For this study, fish larvae were 

 pooled and homogenized to provide adequate repli- 

 cates for methods comparison. On a routine basis, 

 individual larvae may be frozen in 1.5-mL micro- 

 centrifuge vials, then extracted in the same vial, re- 



ducing processing time and errors associated with 

 sample transfer. The actual volumes of 1% sarcosine 

 and TRIS-EDTA buffer may have to be determined 

 empirically depending upon the larval fish size and 

 sample volume required for spectrofluorometric 

 analysis. A trained operator can process approxi- 

 mately 80 samples for RNA and DNA determinations 

 as well as standards in eight hours using CFA and 

 in five hours with FIA. We do not routinely assay 

 replicate sample aliquots or correct for endogenous 

 sample fluorescence when preliminary estimates are 

 less than 3% of total sample fluorescence. 



The "best" method for nucleic acid analysis of lar- 

 val fish may well be determined by sample size, in- 

 strumentation, the presence of interfering sub- 

 stances, or the need to compare values to previously 

 published data. Flow-injection analysis (Caldarone 

 and Buckley, 1991) is a sensitive, precise assay with 

 a simple extraction procedure and high sample 

 throughput. The modified CFA protocol presented 

 here retains those advantages, extends them to a more 

 inexpensive method using static fluorometry, and pro- 

 vides an intercalibration between the two methods. 



one larval fish 



J, 

 add 1% sarcosine in TRIS-EDTA buffer 



i 

 incubate 30 min at room temperature 



i 



vortex vigorously 



J, 



incubate 30 min at room temperature 

 vortex vigorously 



I 



dilute with TRIS-EDTA buffer 



si 

 centrifuge S min at 2,500 x g 



0- 



replicate samples of supernatant 



to spectrofluorometer 



Figure 3 



Generalized flowchart of the sarcosine extraction, flow in- 

 jection analysis (FIA), and conventional fluorometric analy- 

 sis (CFA) procedures. 



