. Author manuscript; available in PMC: 2015 Jul 1.

Published in final edited form as: Horm Behav. 2014 Apr 26;66(2):267–275. doi: 10.1016/j.yhbeh.2014.04.011

Table 5.

Expression stability values (M) for white-throated sparrow samples, as calculated by NormFinder. M values for individual genes and the best combination of two genes in brain, pituitary, ovary, or testis samples were calculated after grouping by plumage morph, breeding stage, or sex.^a

	Morph		Stage		Sex

Tissue	Gene	M	Gene	M	Gene	M
Brain

Best combination	PPIA, TFRC	0.023	PPIA, TFRC	0.023	RPL4, TFRC	0.039

Most stable overall	TFRC	0.027	TFRC	0.028	TFRC	0.044

	PPIA	0.037	PPIA	0.036	RPL4	0.072
	GAPDH	0.044	GAPDH	0.042	GAPDH	0.075
	RPL4	0.050	RPL4	0.050	PPIA	0.076
	RPL32	0.061	RPL32	0.059	18S	0.091
	HPRT	0.071	HPRT	0.070	HPRT	0.117
	18S	0.075	18S	0.076	RPL32	0.121

Pituitary

Best combination	18S, HPRT	0.029	18S, GAPDH	0.018	ACTB, GAPDH	0.041

Most stable overall	GAPDH	0.035	GAPDH	0.017	HPRT	0.053

	18S	0.036	18S	0.032	GAPDH	0.067
	HPRT	0.043	HPRT	0.039	18S	0.067
	ACTB	0.062	ACTB	0.059	HMBS	0.090
	HMBS	0.082	HMBS	0.084	ACTB	0.108

Ovary

Best combination	HMBS, PPIA	0.018	HMBS, PPIA	0.022

Most stable overall	HMBS	0.022	HMBS	0.022

	PPIA	0.029	PPIA	0.028
	GAPDH	0.030	GAPDH	0.030
	RPL4	0.037	RPL4	0.041
	HPRT	0.044	HPRT	0.043
	ACTB	0.062	RPL32	0.059
	RPL32	0.065	ACTB	0.074
	TFRC	0.111	TFRC	0.115
	18S	0.168	18S	0.172

Testis

Best combination	HPRT, PPIA	0.032	HPRT, PPIA	0.027

Most stable overall	HPRT	0.037	HPRT	0.055

	PPIA	0.041	RPL4	0.057
	RPL4	0.057	RPL32	0.065
	RPL32	0.068	PPIA	0.066
	GAPDH	0.082	ACTB	0.099
	TFRC	0.097	TFRC	0.149
	ACTB	0.101	GAPDH	0.153
	HMBS	0.149	HMBS	0.170
	18S	0.185	18S	0.204

Although the rankings changed slightly according to whether the samples were grouped by sex, morph, or stage, the overall patterns were similar. In brain, ranks 2-4 were occupied by PPIA, GAPDH, and RPL4 in all three analyses. In pituitary, GAPDH, 18S, and HPRT were always the top three genes. In ovary, the top five genes were the same regardless of group. In testis, the top four genes were HPRT, PPIA, RPL4, and RPL32. To determine the best combination of two genes, Normfinder uses intergroup stability calculations. Thus, although the top two genes were most often the best combination, other best combinations were sometimes identified, as was the case in pituitary (by sex) and testis (by stage).