RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation

doi:10.4252/wjsc.v16.i6.656

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World J Stem Cells. Jun 26, 2024; 16(6): 656-669
Published online Jun 26, 2024. doi: 10.4252/wjsc.v16.i6.656

Table 1 Candidate reference genes for quantitative real-time polymerase chain reaction

Gene symbol	Function	NCBI access	Primers sequences (5’-3’)	Temperature (°C)
RPLP0	Component of the 60S subunit	NM_001002	F: AGCCCAGAACACTGGTCTC	60
RPLP0	Component of the 60S subunit	NM_001002	R: ACTCAGGATTTCAATGGTGCC	60
TBP	Transcription, metabolic pathways	NM_003194.5	F: CACGAACCACGGCACTGATT	62
TBP	Transcription, metabolic pathways	NM_003194.5	R: TTTTCTTGCTGCCAGTCTGGA	62
YWHAZ	Signal transduction	NM_001135702	F: TGATCCCCAATGCTTCACAAG	61
YWHAZ	Signal transduction	NM_001135702	R: GCCAAGTAACGGTAGTAATCTCC	61
RUNX2	Master transcription factor for osteogenesis	NM_001015051	F: TGGTTACTGTCATGGCGGGTA	62
RUNX2	Master transcription factor for osteogenesis	NM_001015051	R: TCTCAGATCGTTGAACCTTGCTA	62
EF1α	Translation	NM_001402	F: GAAGCTGGTATCTCCAAGAATGG	61
EF1α	Translation	NM_001402	R: CGACAATTAGTTGTTTCACACCC	61
GAPDH	Mitochondrial metabolism	NM_002046	F: GCATCCTGGGCTACACTGA	60
GAPDH	Mitochondrial metabolism	NM_002046	R: CCACCACCCTGTTGCTGTA	60
ACTB	Cytoskeleton	NM_001101	F: CGACAGGATGCAGAAGGAG	60
ACTB	Cytoskeleton	NM_001101	R: TCCTGCTTGCTGATCCACAT	60
B2M	Immune response	NM_004048	F: GAGGCTATCCAGCGTACTCCA	62
B2M	Immune response	NM_004048	R: CGGCAGGCATACTCATCTTTT	62
ALAS1	Mitochondrial metabolism	NM_199166	F: AAATGAATGCCGTGAGGAAAGA	60
ALAS1	Mitochondrial metabolism	NM_199166	R: CCCTCCATCGGTTTTCACACTA	60
TUB	Cytoskeleton	NM_001293212	F: TCAACACCTTCTTCAGTGAAACG	60
TUB	Cytoskeleton	NM_001293212	R: AGTGCCAGTGCGAACTTCATC	60
SDHA	Mitochondrial metabolism	NM_004168	F: CAAACAGGAACCCGAGGTTTT	60
SDHA	Mitochondrial metabolism	NM_004168	R: CAGCTTGGTAACACATGCTGTAT	60

ACTB: Actin beta; ALAS1: Aminolevulinic acid synthase 1; B2M: Beta-2-microglobulin; EF1α: Eukaryotic translational elongation factor 1 alpha; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NCBI: National Cancer for Biotechnology Information: RPLP0: Ribosomal protein, large, P0; RUNX2: Runt-related transcription factor 2; SDHA: Succinate dehydrogenase complex, subunit A, flavoprotein; TBP: TATA-binding protein; TUB: Tubulin; YWHAZ: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta.

Table 2 Gene stability of overall analysis from clonogenic and osteogenic samples

Main group	Rank	Delta Ct		NormFinder		geNorm		BestKeeper
Main group	Rank	Gene	Stability value	Gene	Stability value	Gene	M value	Gene	Std Dev	R	P value
1 to 35 d (clo + ost)	1	TBP	2.13	TBP	1.078	RPLP0/EFα1	0.889	ALAS1	0.70	0.335	0.003
	2	RPLP0	2.27	RPLP0	1.451	-	-	RPLP0	1.33	0.726	0.001
	3	YWAHZ	2.37	YWAHZ	1.62	TBP	1.712	EFα1	1.41	0.668	0.001
	4	EFα1	2.43	ACTB	1.659	YWAHZ	1.923	GAPDH	1.78	0.571	0.001
	5	ACTB	2.43	EFα1	1.707	SDHA	2.037	TBP	1.87	0.857	0.001
	6	ALAS1	2.55	ALAS1	1.84	ALAS1	2.152	ACTB	2.00	0.785	0.001
	7	SDHA	2.57	SDHA	1.948	ACTB	2.261	YWAHZ	2.40	0.876
	8	TUB	2.68	TUB	2.06	B2M	2.337	TUB	2.41	0.777
	9	B2M	2.72	B2M	2.122	TUB	2.402	SDHA	2.59	0.847
	10	GAPDH	2.93	GAPDH	2.385	GAPDH	2.508	B2M	2.63	0.799	0.001

ACTB: Actin beta; ALAS1: Aminolevulinic acid synthase 1; B2M: Beta-2-microglobulin; clo: Clonogenic; EF1α: Eukaryotic translational elongation factor 1 alpha; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ost: Osteogenic; RPLP0: Ribosomal protein, large, P0; SDHA: Succinate dehydrogenase complex, subunit A, flavoprotein; Std Dev: Standard deviation; TBP: TATA-binding protein; TUB: Tubulin; YWHAZ: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta.

Table 3 Gene stability from Subset 1 (clonogenic medium under 35 d) and Subset 2 (osteogenic medium under 35 d)

Subset	Rank	Delta Ct		NormFinder		geNorm		BestKeeper
Subset	Rank	Gene	Stability value	Gene	Stability value	Gene	M value	Gene	Std Dev	R	P value
1: 1 to 35 d (clo)	1	TBP	2.040	TBP	1.024	TBP/YWAHZ	0.996	ALAS1	0.660	0.326	0.043
	2	RPLP0	2.220	RPLP0	1.423	-	-	RPLP0	1.200	0.700	0.001
	3	YWAHZ	2.220	YWAHZ	1.466	B2M	1.319	EFα1	1.250	0.537	0.001
	4	B2M	2.430	B2M	1.790	SDHA	1.449	GAPDH	1.660	0.647	0.001
	5	ACTB	2.460	ACTB	1.818	RPLP0	1.880	TBP	1.900	0.866	0.001
	6	ALAS1	2.480	ALAS1	1.821	EFα1	2.068	ACTB	2.140	0.799	0.001
	7	GAPDH	2.500	GAPDH	1.824	ALAS1	2.159	YWAHZ	2.240	0.850	0.001
	8	EFα1	2.560	EFα1	1.989	GAPDH	2.272	TUB	2.510	0.800	0.001
	9	SDHA	2.620	SDHA	2.099	ACTB	2.356	B2M	2.560	0.855	0.001
	10	TUB	2.680	TUB	2.161	TUB	2.421	SDHA	2.710	0.875	0.001
2: 1 to 35 d (ost)	1	TBP	2.140	TBP	1.087	RPLP0/EFα1	0.515	ALAS1	0.740	0.324	0.044
	2	RPLP0	2.170	RPLP0	1.276	-	-	RPLP0	1.460	0.786	0.001
	3	EFα1	2.180	EFα1	1.291	TBP	1.450	EFα1	1.570	0.812	0.001
	4	ACTB	2.320	ACTB	1.467	YWAHZ	1.777	ACTB	1.720	0.767	0.001
	5	YWAHZ	2.450	YWAHZ	1.758	SDHA	1.860	TBP	1.850	0.846	0.001
	6	SDHA	2.480	SDHA	1.789	ACTB	2.010	GAPDH	1.920	0.519	0.001
	7	TUB	2.570	ALAS1	1.853	TUB	2.108	TUB	2.190	0.744	0.001
	8	ALAS1	2.570	TUB	1.882	ALAS1	2.184	SDHA	2.460	0.818	0.001
	9	B2M	2.970	B2M	2.456	B2M	2.323	YWAHZ	2.530	0.901	0.001
	10	GAPDH	3.270	GAPDH	2.833	GAPDH	2.512	B2M	2.640	0.739	0.001

Table 4 Gene stability from Subset 3 (clonogenic and osteogenic media from 1 to 21 d) and Subset 4 (clonogenic and osteogenic media from 21 to 35 d)

Subset	Rank	Delta Ct		Normfinder		geNorm		BestKeeper
Subset	Rank	Gene	Stability value	Gene	Stability value	Gene	M value	Gene	Std Dev	R	P value
3: 1 to 21 d (clo + ost)	1	TBP	2.160	TBP	1.202	RPLP0/EFα1	0.594	ALAS1	0.750	0.433	0.001
	2	RPLP0	2.230	RPLP0	1.419	-	-	RPLP0	1.100	0.614	0.001
	3	YWAHZ	2.320	ALAS1	1.492	ALAS1	1.207	EFα1	1.100	0.535	0.001
	4	EFα1	2.320	YWAHZ	1.550	TBP	1.639	TBP	1.770	0.801	0.001
	5	ALAS1	2.330	EEF1α1	1.591	YWAHZ	1.814	ACTB	1.940	0.703	0.001
	6	ACTB	2.580	ACTB	1.938	SDHA	1.989	GAPDH	2.010	0.683	0.001
	7	SDHA	2.640	B2M	1.979	B2M	2.104	YWAHZ	2.080	0.819	0.001
	8	B2M	2.640	SDHA	2.048	ACTB	2.275	B2M	2.210	0.709	0.001
	9	TUB	2.790	TUB	2.252	TUB	2.383	SDHA	2.290	0.785	0.001
	10	GAPDH	2.970	GAPDH	2.411	GAPDH	2.499	TUB	2.350	0.738	0.001
4: 21 to 35 d (clo + ost)	1	TBP	1.980	TBP	0.749	RPLP0/EFα1	1.039	ALAS1	0.630	0.345	0.039
	2	ACTB	2.090	ACTB	1.075	-	-	RPLP0	1.740	0.860	0.001
	3	RPLP0	2.120	RPLP0	1.155	ACTB	1.427	EFα1	1.890	0.815	0.001
	4	TUB	2.280	TUB	1.447	TBP	1.636	ACTB	1.920	0.893	0.001
	5	YWAHZ	2.300	YWAHZ	1.604	TUB	1.738	TBP	1.940	0.915	0.001
	6	EFα1	2.360	EEF1α1	1.605	YWAHZ	1.866	GAPDH	1.970	0.456	0.005
	7	SDHA	2.540	SDHA	1.979	SDHA	1.980	TUB	2.120	0.829	0.001
	8	ALAS1	2.670	ALAS1	2.044	B2M	2.087	YWAHZ	2.620	0.899	0.001
	9	B2M	2.760	B2M	2.283	ALAS1	2.210	SDHA	2.810	0.838	0.001
	10	GAPDH	3.420	GAPDH	3.075	GAPDH	2.452	B2M	2.930	0.830	0.001

Table 5 Gene stability from Subset 5 (only osteogenic medium from 1 to 21 d) and Subset 6 (only osteogenic medium from 21 to 35 d)

Subset	Rank	Delta Ct		NormFinder		geNorm		BestKeeper
Subset	Rank	Gene	Stability value	Gene	Stability value	Gene	M value	Gene	Std Dev	R	P value
5: 1 to 21 d (ost)	1	EFα1	2.200	EFα1	1.255	RPLP0/EFα1	0.463	ALAS1	0.810	0.440	0.022
	2	RPLP0	2.210	RPLP0	1.278	-	-	RPLP0	1.330	0.731	0.001
	3	TBP	2.280	TBP	1.325	ACTB	1.514	EFα1	1.330	0.755	0.001
	4	ALAS1	2.380	ALAS1	1.485	TUB	1.629	ACTB	1.540	0.555	0.003
	5	YWAHZ	2.450	YWAHZ	1.706	ALAS1	1.742	TBP	1.860	0.787	0.001
	6	ACTB	2.480	ACTB	1.757	TBP	1.978	TUB	1.880	0.620	0.001
	7	TUB	2.600	TUB	1.948	YWAHZ	2.115	YWAHZ	2.170	0.845	0.001
	8	SDHA	2.660	SDHA	2.043	SDHA	2.222	B2M	2.240	0.609	0.001
	9	B2M	2.950	B2M	2.369	B2M	2.345	SDHA	2.290	0.752	0.001
	10	GAPDH	3.460	GAPDH	3.031	GAPDH	2.568	GAPDH	2.310	0.647	0.001
6: 21 to 35 d (ost)	1	TBP	1.820	TBP	0.578	RPLP0/EFα1	0.495	ALAS1	0.560	0.257	0.304
	2	ACTB	1.900	ACTB	0.584	-	-	TBP	1.750	0.963	0.001
	3	RPLP0	2.050	RPLP0	1.096	ACTB	1.024	RPLP0	1.810	0.878	0.001
	4	EFα1	2.110	EFα1	1.224	TBP	1.134	EFα1	2.040	0.883	0.001
	5	SDHA	2.160	SDHA	1.368	SDHA	1.440	ACTB	2.070	0.963	0.001
	6	YWAHZ	2.300	TUB	1.551	YWAHZ	1.557	SDHA	2.250	0.843	0.001
	7	TUB	2.310	YWAHZ	1.648	TUB	1.655	TUB	2.400	0.856	0.001
	8	ALAS1	2.830	ALAS1	2.254	B2M	1.842	GAPDH	2.510	0.492	0.038
	9	B2M	2.840	B2M	2.401	ALAS1	2.035	YWAHZ	2.760	0.927	0.001
	10	GAPDH	4.040	GAPDH	3.776	GAPDH	2.435	B2M	2.800	0.810	0.001

ACTB: Actin beta; ALAS1: Aminolevulinic acid synthase 1; B2M: Beta-2-microglobulin; EF1α: Eukaryotic translational elongation factor 1 alpha; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ost: Osteogenic; RPLP0: Ribosomal protein, large, P0; SDHA: Succinate dehydrogenase complex, subunit A, flavoprotein; Std Dev: Standard deviation; TBP: TATA-binding protein; TUB: Tubulin; YWHAZ: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta.

Table 6 Gene stability from Subset 7 (only clonogenic medium from 1 to 21 d) and Subset 8 (only clonogenic medium from 21 to 35 d)

Subset	Rank	Delta Ct		NormFinder		geNorm		BestKeeper
Subset	Rank	Gene	Stability value	Gene	Stability value	Gene	M value	Gene	Std Dev	R	P value
7: 1 to 21 d (clo)	1	TBP	2.010	TBP	1.091	RPLP0/EFα1	0.661	ALAS1	0.690	0.379	0.051
	2	RPLP0	2.120	RPLP0	1.379	-	-	EEF1α1	0.820	0.144	0.473
	3	YWAHZ	2.150	YWAHZ	1.398	ALAS1	1.033	RPLP0	0.870	0.516	0.006
	4	ALAS1	2.150	ALAS1	1.404	TBP	1.430	TBP	1.600	0.778	0.001
	5	B2M	2.210	B2M	1.442	YWAHZ	1.617	GAPDH	1.630	0.722	0.001
	6	EFα1	2.370	GAPDH	1.615	B2M	1.718	YWAHZ	1.950	0.759	0.001
	7	GAPDH	2.370	EFα1	1.839	SDHA	1.890	B2M	1.980	0.762	0.001
	8	SDHA	2.610	SDHA	2.089	GAPDH	2.032	ACTB	2.180	0.789	0.001
	9	ACTB	2.630	ACTB	2.133	ACTB	2.212	SDHA	2.320	0.800	0.001
	10	TUB	2.920	TUB	2.546	TUB	2.354	TUB	2.660	0.815	0.001
8: 21 to 35 d (clo)	1	TBP	2.070	TBP	1.063	ACTB/TUB	0.766	ALAS1	0.670	0.480	0.044
	2	RPLP0	2.130	RPLP0	1.247	-	-	GAPDH	1.470	0.455	0.058
	3	TUB	2.200	TUB	1.378	RPLP0	1.417	RPLP0	1.680	0.841	0.001
	4	ACTB	2.210	ACTB	1.430	GAPDH	1.621	EEF1α1	1.780	0.752	0.001
	5	YWAHZ	2.280	YWAHZ	1.601	ALAS1	1.757	ACTB	1.780	0.801	0.001
	6	ALAS1	2.480	ALAS1	1.813	EEF1α1	1.932	TUB	1.850	0.799	0.001
	7	EFα1	2.580	EFα1	1.953	TBP	2.055	TBP	2.170	0.901	0.001
	8	GAPDH	2.680	B2M	2.231	YWAHZ	2.172	YWAHZ	2.520	0.862	0.001
	9	B2M	2.700	GAPDH	2.232	B2M	2.315	B2M	3.070	0.857	0.001
	10	SDHA	2.820	SDHA	2.388	SDHA	2.415	SDHA	3.240	0.884	0.001

ACTB: Actin beta; ALAS1: Aminolevulinic acid synthase 1; B2M: Beta-2-microglobulin; clo: Clonogenic EF1α: Eukaryotic translational elongation factor 1 alpha; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; RPLP0: Ribosomal protein, large, P0; SDHA: Succinate dehydrogenase complex, subunit A, flavoprotein; Std Dev: Standard deviation; TBP: TATA-binding protein; TUB: Tubulin; YWHAZ: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta.

Citation: Ferreira DB, Gasparoni LM, Bronzeri CF, Paiva KBS. RPLP0/TBP are the most stable reference genes for human dental pulp stem cells under osteogenic differentiation. World J Stem Cells 2024; 16(6): 656-669
URL: https://www.wjgnet.com/1948-0210/full/v16/i6/656.htm
DOI: https://dx.doi.org/10.4252/wjsc.v16.i6.656