Validation of reference genes for expression analysis in three Bupleurum species

Figures & data

Table 1. Oligonucleotide primers for amplification of 18 candidate reference genes.

Primer	Sequence^a	Primer	Sequence
UBC6（F）	ACCACTCTTTTTACTTTTTGTTCAT	eIF2a (F）	CAGACTGAGTTGGGTTCTTGTTA
UBC6（R）	TCCACAACTTTTACTATATCCAGATG	eIF2a (R）	TCTGATACTGTTGCCTCTATTCCT
UBC13（F）	CGATGGTGGACTATTTGGGTA	eIF2b	TAATGGGAGCAACTGACCTGT
UBC13（R）	GGCTTTTCTGCTGGTTTGGT	eIF2b	TGGGCAGCAAAGATTGGTAG
ACT7（F）	TCTCTGTGTCTCATATTCTCTCTTTC	eIF3	ACTACGAGGAGGCTGAAAGGT
ACT7（R）	TTCACCATTCCAGTTCCATTG	eIF3	GTTTACCGAGCAGTTTGTGGA
ADF1a（F）	TGCAAGCAACTGATCCAACA	UBC4	ACCAGCCATCGCTTCATCAC
ADF1a（R）	TACAAGAATAACAAGCCCGACA	UBC4（R）	CCCAATCCATCAGACCCACT
ADF1b（F）	CGGCATCTGGTATGGCTG	eIF4	AGACCATTGAACAGCATCCA
ADF1b（R）	CAGGCAGTGAAATCCTCATAGC	eIF4	GAAATGAAATCTACACTAACAACCC
ADF7（F）	TGGGGTTGAGGGCACTTG	ADF5	CGAGTTCCCTCTTGAATCTGTC
ADF7（R）	GACAACATACTACAAGTTCGCCAT	ADF5	TGAGGATTTCACTGCCTGTCTA
ACT1（F）	GTCAAGTGCCCTCAACCCC	ACT2	TGCCCGATGGTCAAGTTAT
ACT1（R）	TGATGTTATTAGAAGTCGTGCCAA	ACT2	GCAGCTTCCATTCCAATCA
ACT4（F）	CCAGAATCAAGCACAATACCAG	eIF6	GTTCAGGCGCAAGGCTTT
ACT4（R）	ACCCAAAAGCAAACAGAGAAAA	eIF6	CTGCAACCGGGTCATTCA
EF1b（F）	TCCTTCTTGTCAACAGCCTTAAT	EF1α	CAACATGATTGAGAGGTCTAGCA
EF1b（R）	AATGGTGATTCTGGGATGGTT	EF1α	GAAGAGGGAGACGAAGAGGTT

^a All sequences are given in the 5′-3′ direction.

Figure 1. Expression levels of candidate reference genes tested using the qPCR cycle threshold values (Cq).

Table 2. Expression levels of 18 reference genes in B. chinense DC., B. falcatum L. and B. scorzonerifolium Willd.

Set^a		ADF1a	eIF2a	EF1b	ACT4	ADF7	eIF3	UBC6	UBC13	ACT7	ACT1	eIF2b	eIF4	ADF1b	UBC4	ADF5	ACT2	eIF6	EF1α
1st set	Mean	16.97	20.21	17.98	21.83	18.23	20.12	27.63	16.96	22.92	18.52	19.86	18.62	16.28	19.86	16.48	16.63	25.70	19.71
	SD	1.11	1.28	1.84	2.00	0.80	1.01	1.25	0.88	1.16	1.20	0.84	1.24	0.70	0.93	0.88	1.10	1.28	1.05
	CV (%)	6.54	6.32	10.26	9.17	4.38	5.03	4.51	5.19	5.05	6.49	4.23	6.67	4.29	4.70	5.35	6.60	4.98	5.34
2nd set	Mean	17.78	20.02	17.89	22.17	18.21	20.49	27.96	15.78	24.55	17.95	18.51	18.44	16.82	20.75	16.72	17.16	39.11	18.58
	SD	1.16	1.55	1.66	1.31	0.45	1.38	2.25	0.79	1.28	1.31	1.03	1.23	0.59	1.05	0.57	0.53	2.77	1.84
	CV (%)	6.53	7.72	9.29	5.93	2.45	6.75	8.04	5.02	5.20	7.30	5.55	6.67	3.50	5.05	3.41	3.10	7.08	9.89
3rd set	Mean	17.86	21.06	18.06	21.61	18.18	20.78	28.26	17.73	22.64	18.95	20.36	19.20	17.41	21.36	17.38	17.48	25.70	19.06
	SD	0.88	0.96	1.66	1.95	0.60	1.54	1.64	1.21	1.12	1.10	1.03	1.24	0.99	1.02	0.60	0.71	1.28	1.60
	CV (%)	4.94	4.57	9.18	9.02	3.29	7.42	5.79	6.82	4.94	5.78	5.06	6.45	5.69	4.79	3.44	4.04	4.98	8.41
4th set	Mean	17.54	20.43	17.98	21.87	18.21	20.46	27.95	16.82	23.37	18.47	19.58	18.75	16.84	20.66	16.86	17.09	30.17	19.12
	SD	1.13	1.36	1.72	1.80	0.63	1.36	1.78	1.27	1.45	1.27	1.25	1.28	0.91	1.18	0.79	0.89	6.60	1.60
	CV (%)	6.46	6.66	9.60	8.22	3.47	6.64	6.36	7.52	6.22	6.89	6.36	6.81	5.38	5.70	4.71	5.19	21.88	8.38

^a The first set to third set consisted of 8 samples of B. chinense DC., B. falcatum L. and B. scorzonerifolium Willd individually. In the fourth set, all three sample sets were included. SD: standard deviation; CV (%): variance coefficient expressed as percentage.

Table 3. Expression stability of 18 candidate reference genes as calculated by GeNorm.

Rank	First set^a		Second set		Third set		Fourth set
	Gene	M values	Gene	M values	Gene	M values	Gene	M values
1	ADF1b	0.301	ACT2	0.391	ADF5	0.325	ADF7	0.394
2	ADF5	0.301	UBC4	0.402	ACT2	0.386	ADF1b	0.394
3	ADF7	0.421	ADF5	0.412	ADF7	0.396	ADF5	0.403
4	eIF2b	0.436	ADF1b	0.452	eIF2b	0.437	ACT2	0.411
5	ACT2	0.506	ADF7	0.496	eIF2a	0.458	UBC4	0.426
6	eIF2a	0.598	UBC13	0.523	ADF1b	0.486	ADF1a	0.454
7	ACT7	0.625	eIF4	0.633	ACT1	0.547	ACT1	0.501
8	eIF3	0.662	ACT7	0.663	ACT7	0.584	ACT7	0.522
9	EF1a	0.693	ADF1a	0.721	ADF1a	0.603	eIF2b	0.572
10	ACT1	0.705	eIF3	0.734	eIF4	0.675	eIF2a	0.687
11	UBC4	0.721	ACT4	0.761	eIF3	0.701	eIF4	0.721
12	ADF1a	0.753	eIF2b	0.781	UBC13	0.746	eIF3	0.743
13	eIF4	0.778	eIF2a	0.799	UBC6	0.785	UBC13	0.833
14	UBC13	0.856	ACT1	0.813	UBC4	0.821	EF1a	0.956
15	UBC6	0.915	EF1a	0.825	EF1a	0.886	UBC6	1.032
16	EF1b	0.956	UBC6	0.907	EF1b	0.904	ACT4	1.085
17	ACT4	1.098	EF1b	0.965	eIF6	1.077	EF1b	1.109
18	eIF6	1.201	eIF6	1.083	ACT4	1.102	eIF4	1.212

^a The expression stability of the first set to the fourth set refer to Table 2.

Table 4. Expression stability of 18 candidate reference genes as calculated by NormFinder.

Rank	First seta		Second set		Third set		Fourth set
	Gene	Stability	Gene	Stability	Gene	Stability	Gene	Stability
1	ADF7	0.237	ADF1b	0.211	ACT7	0.29	ADF1b	0.327
2	ADF1b	0.305	ADF7	0.217	eIF2b	0.37	ADF5	0.334
3	eIF2b	0.327	ADF5	0.349	ADF7	0.377	ACT7	0.434
4	ADF5	0.346	ADF1a	0.332	ACT2	0.395	eIF2b	0.495
5	ACT2	0.349	UBC13	0.441	ADF1b	0.412	ADF7	0.507
6	ACT7	0.367	UBC4	0.444	ADF5	0.451	ADF1a	0.517
7	eIF3	0.411	ACT4	0.582	eIF4	0.52	UBC4	0.552
8	eIF2a	0.444	ACT2	0.587	eIF2a	0.529	eIF2a	0.552
9	ACT1	0.416	eIF4	0.629	ACT1	0.539	ACT1	0.562
10	eIF4	0.484	eIF2a	0.688	UBC4	0.591	eIF4	0.567
11	EF1a	0.513	ACT7	0.693	UBC6	0.611	eIF3	0.587
12	UBC13	0.521	eIF2b	0.701	ADF1a	0.628	ACT2	0.608
13	UBC4	0.531	ACT1	0.744	eIF6	0.653	UBC13	0.691
14	ADF1a	0.628	EF1α	0.746	UBC13	0.682	EF1a	0.727
15	UBC6	0.631	eIF3	0.763	eIF3	0.691	UBC6	0.907
16	ACT4	0.804	EF1b	1.056	EF1b	0.84	ACT4	1.001
17	EF1b	0.828	UBC6	1.077	EF1α	0.852	EF1b	1.053
18	eIF6	0.927	eIF6	1.365	ACT4	0.877	eIF4	1.296

^aThe expression stability of the first set to the fourth set refer to Table 2.

Table 5. Expression stability of 18 candidate reference genes as calculated by BestKeeper.

Rank	First set			Second set			Third set			Fourth set
	Gene	SD	CV	Gene	SD	CV	Gene	SD	CV	Gene	SD	CV
1	ADF7	0.581	3.18	ADF7	0.531	2.16	ADF1b	0.489	3.48	ADF5	0.479	3.72
2	ADF1b	0.612	3.38	ADF5	0.589	2.94	ADF5	0.502	3.99	ADF1b	0.512	3.71
3	ADF5	0.691	4.2	UBC13	0.618	4.33	UBC13	0.511	4.03	ADF7	0.531	2.31
4	eIF2b	0.712	3.56	ADF1b	0.616	4.04	eIF2b	0.654	4.43	UBC13	0.554	4.32
5	UBC13	0.742	4.34	ACT7	0.648	4.62	ADF7	0.746	4.89	ACT2	0.572	4.19
6	ACT7	0.732	4.07	eIF3	0.725	4.26	ACT7	0.758	4.58	ACT7	0.602	4.27
7	eIF3	0.817	3.18	ACT2	0.774	3.53	eIF2a	0.784	4.68	eIF2b	0.616	4.22
8	ACT2	0.816	5.45	eIF2b	0.786	4.18	UBC4	0.793	4.48	eIF2a	0.625	5.43
9	EF1a	0.841	4.27	EF1a	0.868	5.32	eIF3	0.836	4.93	eIF3	0.672	5.31
10	eIF2a	0.853	4.71	UBC4	0.937	3.72	ACT1	0.862	4.97	EF1a	0.737	5.16
11	UBC4	0.892	3.98	ADF1a	0.961	4.02	ACT2	0.925	4.51	eIF4	0.854	4.29
12	ACT1	0.895	4.83	eIF4	1.019	4.52	ADF1a	0.965	5.09	ACT1	0.922	4.45
13	ADF1a	0.928	5.16	ACT1	1.071	4.37	eIF4	1.037	5.48	UBC4	1.232	4.71
14	eIF4	1.023	4.75	ACT4	1.231	5.12	eIF6	1.069	5.65	ADF1a	1.321	5.29
15	ACT4	1.041	5.04	eIF2a	1.341	5.69	UBC6	1.358	5.46	ACT4	1.443	5.94
16	UBC6	1.042	4.77	EF1b	1.332	5.02	EF1a	1.465	5.71	UBC6	1.501	5.31
17	eIF6	1.064	4.93	UBC6	2.095	6.78	EF1b	1.456	5.56	eIF6	1.556	6.76
18	EF1b	1.711	6.52	eIF6	2.335	6.13	ACT4	1.541	5.39	EF1b	1.741	6.28

^a The expression stability of the first set to the fourth set refer to Table 2.

Figure 2. Comparison of the ranking results from BestKeeper, NormFinder and GeNorm. The correlation was evaluated for the ranking results of 18 candidate reference genes in all samples, by Bestkeeper, NormFinder and GeNorm. Correlation coefficient (r) values are shown (*p ＜ 0.05, **p ＜ 0.01).