Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

Table 1 Clinicopathological features of the studied patients n (%)

Clinicopathological feature	Patients with gastric adenocarcinoma	Patients without gastric adenocarcinoma
Age (yr, mean ± SD)	57 ± 15.6	49 ± 14.5
Gender
Male	15 (71)	12 (44)
Female	6 (29)	15 (56)
Location
Cardia	2 (9)	0 (0)
Non-cardia	19 (91)	27 (100)
Histopathological type1
Intestinal	16 (76)	NA
Diffuse	5 (24)	NA
Stage2
Early	4 (19)	NA
Advanced	17 (81)	NA
Tumor Invasion
T1/T2	9 (43)	NA
T3/T4	12 (57)	NA
Lymph node metastasis
Absent	3 (14)	NA
Present	18 (86)	NA
Distant metastasis
Unknown/absent	18 (86)	NA
Present	3 (14)	NA

¹According to the Lauren classification[29];

²according to AJCC[30]. NA: Not applicable.

Table 2 Summary of five reference genes and a target gene

Symbol	Gene name (Assay ID1)	Location	Description	Efficiency
ACTB	β-Actin	7p22	Cytoskeletal structural protein	109%
	(Hs03023943_g1)
B2M	β-2-Microglobulin	15q21	Beta-chain of major histocompatibility complex class I molecules	104%
	(Hs00984230_m1)
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	12p13	Oxidoreductase in glycolysis and gluconeogenesis	102%
	(Hs99999905_m1)
RPL29	Ribosomal protein L29	3p21	Structural constituent of the ribosome	100%
	(Hs00426490_g1)
18S rRNA	18S ribosomal RNA	22p12	Ribosome subunit	110%
	(Hs99999901_s1)
DNMT1	DNA (cytosine-5-)-methyltransferase 1	19p13	Regulation of tissue-specific patterns of methylated cytosine residues	98%
	(Hs00945875_m1)

¹TaqMan^® probes were purchased as Assays-on-Demand Products for Gene Expression (Life Technologies, Foster City, CA).

Table 3 Ranking of the candidate single reference genes by each software package used

	NormFinder		geNorm™		BestKeeper		DataAssist™
	Stability value1	Ranking	M value1	Ranking	Coefficient of variance1	Ranking	Score1	Ranking
Neoplastic tissues
	0.24	ACTB	0.07	GAPDH	9.78	ACTB	1.58	ACTB
	0.73	B2M	0.07	B2M	11.77	B2M	1.70	B2M
	0.99	GAPDH	0.08	ACTB	13.39	GAPDH	1.88	GAPDH
	1.41	18S rRNA	0.12	18S rRNA	17.14	18S rRNA	2.25	18S rRNA
Adjacent non-neoplastic tissues
	0.61	B2M	0.05	GAPDH	7.80	ACTB	1.65	B2M
	0.65	GAPDH	0.05	B2M	9.61	B2M	1.77	ACTB
	0.65	ACTB	0.07	ACTB	10.52	GAPDH	1.81	GAPDH
	1.53	18S rRNA	0.13	18S rRNA	16.57	18S rRNA	2.46	18S rRNA
Matched pairs of adjacent non-neoplastic and neoplastic gastric tissues
	0.14	ACTB	0.06	GAPDH	9.24	ACTB	1.83	GAPDH
	0.29	B2M	0.06	B2M	11.31	B2M	1.83	ACTB
	0.41	GAPDH	0.07	ACTB	12.52	GAPDH	2.00	B2M
	0.54	18S rRNA	0.13	18S rRNA	16.86	18S rRNA	2.60	18S rRNA
Normal gastric tissues
	0.38	GAPDH	0.06	GAPDH	7.38	18S rRNA	1.82	GAPDH
	0.41	ACTB	0.06	ACTB	11.08	GAPDH	2.24	ACTB
	1.07	B2M	0.07	B2M	11.46	ACTB	2.40	B2M
	2.24	18S rRNA	0.11	18S rRNA	13.34	B2M	3.91	18S rRNA
All gastric tissues
	0.14	ACTB	0.07	GAPDH	10.89	ACTB	2.04	ACTB
	0.36	B2M	0.07	B2M	13.12	B2M	2.09	B2M
	0.69	GAPDH	0.08	ACTB	13.24	18S rRNA	2.12	GAPDH
	0.96	18S rRNA	0.13	18S rRNA	13.43	GAPDH	3.34	18S rRNA
Cell lines
	0.53	ACTB	0.06	ACTB	2.36	18S rRNA	1.71	ACTB
	1.55	B2M	0.06	B2M	4.81	ACTB	2.45	B2M
	1.73	18S rRNA	0.13	18S rRNA	8.60	B2M	2.63	18S rRNA
All gastric tissues + cell lines
	0.45	ACTB	0.09	ACTB	10.67	ACTB	2.73	B2M
	1.11	B2M	0.09	B2M	13.02	B2M	3.32	ACTB
	1.19	18S rRNA	0.16	18S rRNA	13.41	18S rRNA	3.38	18S rRNA

¹A lower value indicates increased stability in gene expression.

Table 4 Best reference gene combinations according to each software

Neoplastic tissues	Adjacentnon-neoplastic tissues	Normal tissues	Matched pairs of adjacent nonneoplastic and neoplastic gastric tissues	All gastric tissues	Cell lines	All gastric tissues+cell lines
ACTB + B2M	GAPDH + B2M	GAPDH + ACTB	ACTB + B2M	ACTB + B2M	ACTB + B2M	ACTB + 18S rRNA
GAPDH + B2M	GAPDH + B2M	GAPDH + ACTB	GAPDH + B2M	GAPDH + B2M	ACTB + B2M	ACTB + B2M
GAPDH + B2M	GAPDH + B2M	GAPDH + B2M	GAPDH + B2M	GAPDH + B2M	ACTB + B2M	ACTB + B2M
ACTB + B2M	GAPDH + B2M	GAPDH + B2M	GAPDH + B2M	GAPDH + B2M	ACTB + B2M	ACTB + B2M