The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale

Table 3 Pair-wise statistical analyses of the inter-group stability of selected reference genes.

	Group comparisons	Yellow vs 11-KT		Yellow vs Silver		11-KT vs silver
Tissue	Reference genes	p based on REST 2009	p based on Mann-Whitney	p based on REST 2009	p based on Mann-Whitney	p based on REST 2009	p based on Mann-Whitney
2009
Liver	*eef1* - *18 s* ( *GeNorm* )	0.898	0.815	0.071	0.074	0.067	0.190
	eef1-l36 (GeNorm)	0.457	0.481	0.115	0.167	0.012	0.031
	18 s (BestKeeper, Normfinder)	0.085	0.093	< 0.001	0.001	0.243	0.297
	*eef1* ( *BestKeeper* )	0.187	0.114	0.651	1.000	0.060	0.094
	nop14 (NormFinder)	0.224	0.321	0.040	0.059	0.105	0.387
	actb-l36 (Normfinder)	0.002	0.002	< 0.001	< 0.001	0.238	0.297
Ovary	eef1-actb (GeNorm)	0.704	0.073	0.281	0.093	0.525	0.546
	*l36* ( *BestKeeper* )	0.533	0.673	0.638	0.673	0.784	0.863
	nop14 (NormFinder)	0.850	0.963	0.007	0.011	0.030	0.050
	nop14-l36 (NormFinder)	0.899	0.673	0.079	0.139	0.135	0.136
2008
Liver	*eef1* - *18 s* (2009 best)	0.082	0.079	0.892	1.000	0.166	0.113
	eef1 (2009 2^nd best)	0.002	0.004	0.283	0.356	0.011	0.019
	eef1-l36 (GeNorm)	0.006	0.006	0.869	0.905	0.006	0.006
	*18 s* ( *BestKeeper* )*	0.308	0.549	0.422	0.053	0.884	0.063
	nop14 (Normfinder)	0.047	0.053	0.038	0.035	0.001	0.003
	nop14-actb (Normfinder)	0.343	0.549	0.002	0.003	0.027	0.024
Ovary	*l36* (2009 best)	0.307	0.321	0.924	0.730	0.321	0.298

* 18 s can only be considered the best for 2008 liver with qualifications as Mann-Whitney test showed significant difference between Yellow and Silver groups.
The top ranked candidate reference genes as selected by GeNorm, BestKeeper and NormFinder were validated using REST 2009 (QIAGEN 2009; see text for explanation) and Mann-Whitney. To reduce Type II error, differences are considered significant at p ≤ 0.055 (see text for further explanation). Significant difference between any two groups indicates unsuitability as reference genes due to lack of stability across groups. Figures in bold denote the best reference gene(s) based on inter-group stability. The second best option is also denoted in bold when the best included 18 s (e.g, liver). The best or second best reference gene(s) were applied to the 2008 data. Where it performs poorly (e.g., eef1 liver data), the programs were used to select the top ranked candidate gene based on 2008 data, with the statistical validation of the results also shown above.

ISSN: 1471-2199