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tissue was frozen at -80°C, indicating that DNA was 
stable during that time and under that condition. 
Neither freezing and thawing nor storage of frozen 
tissue affected RNA in muscle cell nuclei of larvae of 
Walleye Pollock in our study. For frozen human tissue, 
no significant RNA degradation (as measured by gene 
expression and electropherograms) was detected after 
16 h on ice (Micke et ah, 2006), and another study 
showed that significant RNA degradation did not be- 
gin until 30 min after thawing at room temperature 
(Botling et al., 2009). Although those studies did not 
measure the amount of RNA present, they do support 
our assertion that the protocol used in our study was 
adequate to preserve RNA because larvae were frozen 
quickly, thawed tissue was kept cool on ice, and the 
time from tissue thawing to analysis with flow cytom- 
etry was typically not longer than 5 h. Our results dif- 
fer from the findings of Theilacker and Shen (1993b) 
in that their study indicated that a cryoprotectant and 
acid were needed before freezing to stabilize RNA in 
brain cells of larvae of Walleye Pollock. This difference 
in results may be due to the difference in type of tissue 
used (muscle cell nuclei versus whole brain cells) (Oli- 
var et al., 2009) or in the method used for preparation 
of tissue for flow cytometry. In Theilacker and Shen 
(1993b), tissue dissection occurred before freezing and 
whole cells were analyzed; however, in our study, tis- 
sue preparation occurred after freezing, and only nuclei 
were used. 
Our results indicate that only frozen tissue should 
be analyzed when condition is measured with the 
method described here. Results would be inaccurate if 
fresh tissue were used because of its higher fraction of 
S-phase nuclei. Cell-cycle measurements (fraction of S- 
and G2/M-phase nuclei pooled) of independent groups 
of larvae of Walleye Pollock reared under similar condi- 
tions were not significantly different (Porter and Bailey, 
2011), indicating that the effect of freezing and thaw- 
ing was consistent. Freezing and thawing of tissue was 
also part of the method used in another study where 
standardized protocols were used for spectrofluoromet- 
ric analysis of RNA and DNA for assessing larval fish 
physiological condition (Caldarone et al., 2006). 
Conclusions 
An nRNA covariate improved larval condition measure- 
ments. For minimal cost (the cost of the Syto RNAS- 
elect stain), model accuracy increased, and the greatest 
improvement was for small larvae. The assay that we 
developed in our study quickly determines condition, 
and, therefore, many larvae can be analyzed in a short 
period of time. In addition, larvae can be kept frozen 
for at least 10 months without affecting condition 
measurements. There is no diel pattern in RNA con- 
tent or measurements of the cell-cycle phases for lar- 
vae of Walleye Pollock (Bailey et al., 1995; Theilacker 
and Shen, 2001); therefore, our method can be applied 
in the field, where sampling can occur anytime during 
a 24-h period. Because we found that temperature af- 
fected RSG1, future studies should include measure- 
ments at additional temperatures to formulate a model 
for field-sampled larvae. 
Acknowledgments 
We would like to thank A. Dougherty for collection of 
Walleye Pollock eggs and her assistance in the labo- 
ratory. D. Prunkard at the University of Washington, 
Department of Pathology, Cytometry Core Facility as- 
sisted with flow cytometry. F. Morado, M. Paquin and 
3 anonymous reviewers provided helpful comments on 
earlier drafts of the manuscript. This research was 
funded by the North Pacific Research Board (NPRB 
grant no. 926, publication 432) and the Alaska Fisher- 
ies Science Center. It is contribution EcoFOCI-0800 to 
NOAA’s Ecosystems and Fisheries-Oceanography Coor- 
dinated Investigations. 
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