Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

Table 2

The evaluation of candidate reference genes with NormFinder and BestKeeper¹.

Method		Ranking order (better-good-average)
	Rank^1	1	2	3	4	5	6	7	8	9	10
	Organs/seedlings^2
NormFinder	Gene^3	ARF	EF1α	COX	GAPDH	RPL2	Actin	H3	18S rRNA	Tubα	CYP
	M1	0.131	0.203	0.365	0.373	0.382	0.389	0.427	0.582	0.678	0.703
BestKeeper	Gene^3	18S rRNA	CYP	COX	Actin	RPL2	ARF	EF1α	H3	GAPDH	Tubα
	SD	0.58	0.72	0.94	1.07	1.07	1.08	1.10	1.13	1.28	1.58
	Heat
NormFinder	Gene^3	ARF	RPL2	EF1α	CYP	H3	GAPDH	COX	18S rRNA	Actin	Tubα
	M1	0.212	0.228	0.238	0.387	0.424	0.454	0.500	0.757	0.850	0.928
BestKeeper	Gene^3	Actin	18S rRNA	GAPDH	RPL2	ARF	H3	EF1α	CYP	COX	Tubα
	SD	0.57	0.60	0.77	0.92	0.93	0.95	1.10	1.11	1.16	1.67
	CdCl2
NormFinder	Gene^3	CYP	RPL2	ARF	EF1α	H3	GAPDH	COX	Actin	18S rRNA	Tubα
	M1	0.625	0.65	0.75	0.751	0.765	0.791	0.836	0.91	0.987	1.621
BestKeeper	Gene^3	ARF	EF1α	CYP	Actin	18S rRNA	RPL2	H3	GAPDH	COX	Tubα
	SD	0.37	0.49	0.53	0.54	0.62	0.98	1.05	1.22	1.33	2.09
	NaCl
NormFinder	Gene^3	EF1α	ARF	CYP	GAPDH	H3	COX	Actin	18S rRNA	RPL2	Tubα
	M1	0.201	0.213	0.251	0.275	0.304	0.324	0.426	0.492	0.496	0.852
BestKeeper	Gene^3	18S rRNA	ARF	COX	EF1α	Actin	GAPDH	CYP	H3	RPL2	Tubα
	SD	0.24	0.32	0.34	0.38	0.39	0.45	0.61	0.66	0.86	1.24
	NaHCO3
NormFinder	Gene^3	RPL2	Actin	CYP	GAPDH	H3	COX	ARF	EF1α	Tubα	18S rRNA
	M1	0.253	0.454	0.48	0.485	0.489	0.57	0.832	1.184	1.273	1.407
BestKeeper	Gene^3	Actin	RPL2	H3	GAPDH	COX	CYP	18S rRNA	ARF	EF1α	Tubα
	SD	0.64	0.74	0.86	0.89	1.02	1.06	1.11	1.22	1.45	2.13
	Na2CO3
NormFinder	Gene^3	ARF	H3	RPL2	CYP	EF1α	Actin	Tubα	18S rRNA	COX	GAPDH
	M1	0.284	0.297	0.298	0.366	0.413	0.432	0.455	0.469	0.473	0.523
BestKeeper	Gene^3	COX	Actin	ARF	GAPDH	H3	Tubα	CYP	RPL2	EF1α	18S rRNA
	SD	0.36	0.38	0.68	0.7	0.76	0.85	0.89	0.89	0.92	1.03

¹Lower stability value M1 obtained by NormFinder or SD obtained by BestKeeper indicates higher stability of housekeeping genes under each condition and is marked with bold text. ²These samples include leaves, stems, roots, flowers, and seedlings, as well as the leaves and roots under heat (37°C for 0, 1, 2, 4, 6, 8, 12, 24, and 48 h), heavy metal (200 μM CdCl₂ for 0, 1, 3, 6, 12, 24, and 48 h), salt (200 mM NaCl for 0, 1, 3, 6, 12, 24, and 48 h), and alkali (200 mM NaHCO₃ or 100 mM Na₂CO₃, treated for 0, 1, 3, 6, 12, 24, and 48 h) treatments. Three biological replicates were taken for different organs/seedlings, as well as leaves and roots at each time point of all stress treatments. ³Names of candidate reference genes refer to Table 1.