Validation of reference genes in a wide of samples Functional Plant Biology 347 Table 3. Expression stability values of reference genes ranked by geNorm and NormFinder for the four strategic groups and all 20 samples together (ranking in parentheses) Gene geNorm NormFinder Total Developmental Different Biotic Abiotic Total Developmental Different Biotic Abiotic stages tissues stress stress stages tissues stress stress ACTIN 0.61(1) 0.12(1) 0.56(4) 0.01(1) 0.46(2) 0.09(1) 0.20(3) 0.17(1) 0.05(5 0.26(3) ACTIN-I 1.21(8) 0.36(5) 0.52(3) 0.71(12) 0.51(3) 0.90(9) 0.34(7 0.463) 0.74(13) 0.32(5) ACTIN-2 1.11(7 0.53(7 0.82(7 0.43(8) 0.65(7 0.85(8) 0.33(6 0.56(6 0.40(7) 0.57(10) GAPDH 1.02(6) 0.20(2) 1.03(9) 0.31(7) 0.70(8) 0.68(5) 0.29(5) 0.69(9) 0.56(10) 0.50(8) CyP 0.61(1) 0.12(1) 0.45(2) 0.12(5 0.54(4) 0.50(4) 0.05(1) 0.41(2) 0.02(3) 0.32(6 EFI-a 0.74(3) 0.23(3) 0.66(5) 0.22(6) 0.281) 0.39(2) 0.28(4) 0.51(4) 0.40(8) 0.32(4) Tub a 0.92(5) 0.77(9) 0.31(1) 0.01(1) 0.60(6 0.85(7) 0.87(11) 0.53(5) 0.04(4) 0.34(7) 18S rRNA 1.43(10) 1.54(12) 1.22(11) 0.58(10) 0.57(5) 1.17(12) 3.44(13) 1.03(12) 0.62(11) 0.18(1) UBC30 0.72(2) 0.34(4) 0.31(1) 0.061(2) 0.28(1) 0.46(3) 0.52(8) 0.62(8) 0.00(1) 0.21(2) UBO 0.86(4) 0.66(8) 0.91(8) 0.07(3) 0.74(9) 0.79(6 0.59(9) 0.81(10) 0.00(2) 0.54(9) PPR 1.31(9) 0.84(10) 0.77(6 0.08(4) 0.95(12) 1.05(10 0.97(12) 0.60(7) 0.11(6) 0.91(13) PP2A 1.90(12) 0.44(6) 1.54(12) 0.50(9) 0.80(10) 2.51(13) 0.05(2) 2.21(13) 0.47(9) 0.68(11) MDH 1.56(11) 0.92(11) 1.12(10) 0.65(11) 0.86(11) 1.16(11) 0.68(10) 0.83(11) 0.73(12) 0.68(12) GAPDH,EFI-o.and UBC30.Furthermore,the genes ACTIN. for accurate normalisation,i.e.ACTIN,CyP,UBC30,EFI-o and CyP and GAPDH with lowest M values displayed less variation UBO.When comparing developmental stages,ACTIN and Cyp (from 0.I to 0.3)than M values ofother genes.UBC30 and Tub_a formed the optimal set of reference genes,whereas in different were the most stable genes when gene expression in different tissue samples,UBC30,Tub a and CyP were considered as the tissue samples was compared,followed by Cyp.For the biotic most suitable set ofreference genes.CyP and Tub a were the most stress treated samples,CyP and Tub_a showed lowest M values. stable reference genes in the biotic stress samples.For the abiotic The UBC30 and EF/-o genes displayed highest stability under stress treated samples,the gene set UBC30.EFI-a and ACTIN abiotic stress conditions,followed by ACTIN. was selected for most optimal normalisation.Generally,these The geNorm software was also used to calculate the pair-wise results show that different experiments (here comparing variation(Vn/Vn+1)for the determination of the optimal number developmental stages,tissues or stress treatments)require sets of control genes between the sequential normalisation factors of different reference genes for normalisation. (NF)(NFn and NFn+1)(Fig.1).Data obtained from all 20 samples were analysed together and showed that the V value with the inclusion of a fifth gene (V5/6)was 0.146,which was lower NormFinder analysis than the arbitrary cut off value of 0.15.This means that the most The NormFinder program was used as a different means for suitable set of reference genes should contain at least five genes further validation of the data.There were clear differences in 0.45 V2/3 ▣V3/4 ☐V4/5 ▣V56 0.40 ■V6/7 ☐V7/8 ☐V8/9 mV9/10 题V10W11 目V11/12 ▣V1213 0.35 0.30 0.25 0.20 0.15 0.10 0.05 Total Develoomental Different tissues Biotic stress Abiotic stress stages Fig.1.Pairwise variation(V)measureofthe candidate reference genes.When Vn/Vn +1 <0.15,then the optima number of reference genes is N.The optimal positions are indicated with an asterisk for the four strategic groups and all 20 samples together.GAPDH, EF1-a and UBC30. Furthermore, the genes ACTIN, CyP and GAPDH with lowest M values displayed less variation (from 0.1 to 0.3) than M values of other genes. UBC30 and Tub_a were the most stable genes when gene expression in different tissue samples was compared, followed by CyP. For the biotic stress treated samples, CyP and Tub_a showed lowest M values. The UBC30 and EF1-a genes displayed highest stability under abiotic stress conditions, followed by ACTIN. The geNorm software was also used to calculate the pair-wise variation (Vn/Vn + 1) for the determination of the optimal number of control genes between the sequential normalisation factors (NF) (NFn and NFn + 1) (Fig. 1). Data obtained from all 20 samples were analysed together and showed that the V value with the inclusion of a fifth gene (V5/6) was 0.146, which was lower than the arbitrary cut off value of 0.15. This means that the most suitable set of reference genes should contain at least five genes for accurate normalisation, i.e. ACTIN, CyP, UBC30, EF1-a and UBQ. When comparing developmental stages, ACTIN and CyP formed the optimal set of reference genes, whereas in different tissue samples, UBC30, Tub_a and CyP were considered as the most suitable set of reference genes.CyPandTub_awerethe most stable reference genes in the biotic stress samples. For the abiotic stress treated samples, the gene set UBC30, EF1-a and ACTIN was selected for most optimal normalisation. Generally, these results show that different experiments (here comparing developmental stages, tissues or stress treatments) require sets of different reference genes for normalisation. NormFinder analysis The NormFinder program was used as a different means for further validation of the data. There were clear differences in Table 3. Expression stability values of reference genes ranked by geNorm and NormFinder for the four strategic groups and all 20 samples together (ranking in parentheses) Gene geNorm NormFinder Total Developmental stages Different tissues Biotic stress Abiotic stress Total Developmental stages Different tissues Biotic stress Abiotic stress ACTIN 0.61 (1) 0.12 (1) 0.56 (4) 0.01 (1) 0.46 (2) 0.09 (1) 0.20 (3) 0.17 (1) 0.05 (5) 0.26 (3) ACTIN-1 1.21 (8) 0.36 (5) 0.52 (3) 0.71 (12) 0.51 (3) 0.90 (9) 0.34 (7) 0.46 (3) 0.74 (13) 0.32 (5) ACTIN-2 1.11 (7) 0.53 (7) 0.82 (7) 0.43 (8) 0.65 (7) 0.85 (8) 0.33 (6) 0.56 (6) 0.40 (7) 0.57 (10) GAPDH 1.02 (6) 0.20 (2) 1.03 (9) 0.31 (7) 0.70 (8) 0.68 (5) 0.29 (5) 0.69 (9) 0.56 (10) 0.50 (8) CyP 0.61 (1) 0.12 (1) 0.45 (2) 0.12 (5) 0.54 (4) 0.50 (4) 0.05 (1) 0.41 (2) 0.02 (3) 0.32 (6) EF1-a 0.74 (3) 0.23 (3) 0.66 (5) 0.22 (6) 0.28 (1) 0.39 (2) 0.28 (4) 0.51 (4) 0.40 (8) 0.32 (4) Tub_a 0.92 (5) 0.77 (9) 0.31 (1) 0.01 (1) 0.60 (6) 0.85 (7) 0.87 (11) 0.53 (5) 0.04 (4) 0.34 (7) 18S rRNA 1.43 (10) 1.54 (12) 1.22 (11) 0.58 (10) 0.57 (5) 1.17 (12) 3.44 (13) 1.03 (12) 0.62 (11) 0.18 (1) UBC30 0.72 (2) 0.34 (4) 0.31 (1) 0.061 (2) 0.28 (1) 0.46 (3) 0.52 (8) 0.62 (8) 0.00 (1) 0.21 (2) UBQ 0.86 (4) 0.66 (8) 0.91 (8) 0.07 (3) 0.74 (9) 0.79 (6) 0.59 (9) 0.81 (10) 0.00 (2) 0.54 (9) PPR 1.31 (9) 0.84 (10) 0.77 (6) 0.08 (4) 0.95 (12) 1.05 (10) 0.97 (12) 0.60 (7) 0.11 (6) 0.91 (13) PP2A 1.90 (12) 0.44 (6) 1.54 (12) 0.50 (9) 0.80 (10) 2.51 (13) 0.05 (2) 2.21 (13) 0.47 (9) 0.68 (11) MDH 1.56 (11) 0.92 (11) 1.12 (10) 0.65 (11) 0.86 (11) 1.16 (11) 0.68 (10) 0.83 (11) 0.73 (12) 0.68 (12) 0.45 Pairwise variation (V) V2/3 V6/7 V10/11 V3/4 V7/8 V11/12 V4/5 V8/9 V12/13 V5/6 0.40 V9/10 0.35 0.30 0.25 * * * * * 0.20 0.15 0.10 0.05 0 Total Developmental stages Different tissues Biotic stress Abiotic stress Fig. 1. Pairwise variation (V) measure of the candidate reference genes.When Vn/Vn + 1 <0.15,then the optimal number of reference genes is N. The optimal positions are indicated with an asterisk for the four strategic groups and all 20 samples together. Validation of reference genes in a wide of samples Functional Plant Biology 347