Selection and validation of reference gene for RT-qPCR studies in co-culture system of mouse cementoblasts and periodontal ligament cells

Yong, Jiawen; Groeger, Sabine; Ruiz-Heiland, Gisela; Ruf, Sabine

doi:10.1186/s13104-022-05948-x

Table 1 The rank of candidate reference gene stability for monocultured OCCM-30 or SV-PDL, direct cell–cell culture, co-cultured OCCM-30 cells and co-cultured SV-PDL cells, overall ranks were calculated by the four algorithms application (geNorm, NormFinder, comparative ΔC_q and BestKeeper, respectively)

From: Selection and validation of reference gene for RT-qPCR studies in co-culture system of mouse cementoblasts and periodontal ligament cells

Rank	Methods		geNorm		NormFinder		Comparative ΔC_q		BestKeeper
Rank	Ranking order	Ranking sum	Ranking order	Stability value (M)	Ranking order	Stability value	Ranking order	Stability value	Ranking order	Stability value (r)	SD (± C_q)	CV (% C_q)
Monocultured cementoblasts (OCCM-30)
(1)	RPL22	13	GUSB	0.032	TBP	0.065	RPL22	0.22	PPIB	0.750	0.470	2.278
(2)	PPIB	17	POLR2A	0.037	PPIB	0.065	RPLP0	0.26	YWHAZ	0.685	1.541	7.321
(3)	POLR2A	21	RPL22	0.042	β-actin	0.092	POLR2A	0.40	GAPDH	0.679	0.390	2.282
(4)	RPLP0	23	RPLP0	0.058	RPL22	0.136	GUSB	0.40	TBP	0.620	0.503	2.121
(5)	GUSB	23	UBC	0.063	RPL	0.212	UBC	0.53	RPL22	0.612	0.662	2.679
(6)	β-actin	24	β-actin	0.072	RPLP0	0.538	PPIB	0.54	β-actin	0.453	0.543	3.208
(7)	TBP	24	GAPDH	0.083	POLR2A	0.719	EEF1A1	0.59	RPL	0.172	0.204	0.790
(8)	UBC	27	PPIB	0.090	GUSB	0.831	TBP	0.59	UBC	-0.038	0.489	1.756
(9)	GAPDH	32	YWHAZ	0.095	UBC	0.859	β-actin	0.64	POLR2A	-0.115	0.197	0.875
(10)	YWHAZ	35	EEF1A1	0.113	EEF1A1	1.005	RPL	0.81	GUSB	-0.282	0.331	1.393
(11)	EEF1A1	39	TBP	0.183	GAPDH	2.421	GAPDH	2.12	RPLP0	-0.428	0.152	0.988
(12)	RPL	-			YWHAZ	2.461	YWHAZ	2.29	EEF1A1	-0.683	0.472	2.774
(13)	TUBB	-					TUBB	13.85	TUBB	-0.986	0.295	0.795
Monocultured periodontal ligament cells (SV-PDL)
(1)	GUSB	14	GUSB	0.003	EEF1A1	0.112	GUSB	0.18	TBP	0.928	0.447	1.766
(2)	RPLP0	19	GAPDH	0.004	GUSB	0.112	RPLP0	0.19	RPL22	0.921	0.762	2.951
(3)	RPL22	22	RPLP0	0.007	POLR2A	0.123	EEF1A1	0.19	UBC	0.916	0.660	2.360
(4)	TBP	22	β-actin	0.021	β-actin	0.124	POLR2A	0.24	YWHAZ	0.910	0.455	2.103
(5)	β-actin	22	PPIB	0.031	TBP	0.124	RPL22	0.26	β-actin	0.905	0.537	3.093
(6)	PPIB	26	RPL22	0.040	RPLP0	0.125	TUBB	0.31	GAPDH	0.868	0.518	2.894
(7)	POLR2A	27	UBC	0.045	PPIB	0.206	PPIB	0.51	PPIB	0.739	0.446	2.094
(8)	GAPDH	30	TBP	0.050	TUBB	0.282	TBP	0.53	RPLP0	0.440	0.126	0.818
(9)	TUBB	36	POLR2A	0.075	RPL22	0.475	β-actin	0.66	RPL	0.355	0.250	0.996
(10)	EEF1A1	38	TUBB	0.100	RPL	0.526	RPL	0.92	GUSB	0.328	0.138	0.561
(11)	YWHAZ	49	EEF1A1	0.127	GAPDH	5.241	GAPDH	4.98	POLR2A	0.169	0.226	0.989
(12)	UBC	-	YWHAZ	0.850	YWHAZ	6.351	YWHAZ	5.99	TUBB	0.125	0.268	0.930
(13)	RPL	-					UBC	10.46	EEF1A1	-0.292	0.168	0.967
Direct cell–cell contact cultured of OCCM-30 and SV-PDL
(1)	GUSB	7	POLR2A	0.086	GUSB	0.005	RPLP0	0.29	POLR2A	0.912	0.57	2.27
(2)	POLR2A	8	GUSB	0.089	POLR2A	0.007	GUSB	0.48	GUSB	0.889	0.62	2.36
(3)	RPLP0	14	RPL22	0.11	RPL22	0.019	PPIB	0.48	PPIB	0.872	1.07	4.83
(4)	RPL22	15	RPLP0	0.212	RPLP0	0.029	POLR2A	0.59	RPL22	0.784	0.62	2.22
(5)	PPIB	16	PPIB	0.399	PPIB	0.058	RPL22	1.09	RPLP0	0.575	0.31	2.00
Co-cultured OCCM-30 control
(1)	PPIB	8	PPIB	0.211	GUSB	0.006	RPLP0	0.31	RPL22	0.986	1.5	5.31
(2)	GUSB	10	POLR2A	0.224	PPIB	0.013	GUSB	1.00	PPIB	0.977	0.95	4.34
(3)	RPLP0	14	RPLP0	0.325	RPL22	0.028	PPIB	1.02	GUSB	0.977	0.94	3.46
(4)	POLR2A	14	GUSB	0.347	POLR2A	0.040	POLR2A	1.03	POLR2A	0.658	1.01	3.92
(5)	RPL22	14	RPL22	0.704	RPLP0	0.045	RPL22	1.59	RPLP0	0.220	0.26	1.65
Co-cultured OCCM-30 with 3 indicated time (n = 18)
(1)	PPIB	9	RPLP0	0.409	PPIB	0.016	RPLP0	0.63	PPIB	0.949	0.72	3.25
(2)	GUSB	9	GUSB	0.478	GUSB	0.021	POLR2A	0.74	GUSB	0.767	0.6	2.18
(3)	RPLP0	11	PPIB	0.515	POLR2A	0.027	GUSB	0.78	RPL22	0.712	0.98	3.54
(4)	POLR2A	14	POLR2A	0.565	RPL22	0.031	PPIB	0.78	RPLP0	0.500	0.49	2.9
(5)	RPL22	17	RPL22	0.840	RPLP0	0.035	RPL22	1.27	POLR2A	0.427	0.64	2.48
Co-cultured SV-PDL control
(1)	PPIB	9	RPLP0	0.308	PPIB	0.004	RPLP0	0.11	POLR2A	0.998	0.65	2.59
(2)	POLR2A	11	PPIB	0.309	RPL22	0.004	PPIB	0.52	RPL22	0.983	0.68	2.59
(3)	RPLP0	12	POLR2A	0.378	POLR2A	0.009	RPL22	0.62	GUSB	0.980	0.53	1.85
(4)	RPL22	12	GUSB	0.390	GUSB	0.011	POLR2A	0.74	PPIB	0.973	0.47	2.17
(5)	GUSB	16	RPL22	0.732	RPLP0	0.023	GUSB	0.78	RPLP0	0.700	0.09	0.61
Co-cultured SV-PDL with 3 indicated time (n = 18)
(1)	PPIB	8	POLR2A	0.161	PPIB	0.005	RPLP0	0.22	PPIB	0.980	0.31	1.44
(2)	POLR2A	10	GUSB	0.165	RPLP0	0.015	PPIB	0.42	POLR2A	0.823	0.5	1.98
(3)	RPLP0	11	RPLP0	0.214	GUSB	0.016	POLR2A	0.60	GUSB	0.774	0.54	2.04
(4)	GUSB	12	PPIB	0.218	POLR2A	0.017	GUSB	0.60	RPL22	0.746	0.57	2.02
(5)	RPL22	19	RPL22	0.422	RPL22	0.019	RPL22	0.74	RPLP0	0.646	0.19	1.21

A higher rank denotes lower expression stability. Individual primer efficiency was taken in to account by the abbreviations of C_q (threshold cycle), SD (standard deviation), CV (coefficient of variation) and r (Pearson’s correlation coefficient). The genes are ordered form the highest to the lowest ranking

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ISSN: 1756-0500

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