Screening of Reference Genes for RT-qPCR in Neoscytalidium dimidiatum

doi:10.13560/j.cnki.biotech.bull.1985.2022-1322

Abstract

Abstract:

The pitaya canker disease is a serious fungal disease caused by Neoscytalidium dimidiatum. In recent years, the outbreak of the pitaya canker disease in the world's pitaya producing areas has seriously affected the pitaya industry development. In order to study the expression level and function of genes responding to stress in N. dimidiatum, it is necessary to screen the reference genes that express stably when the pathogen grows under different cultural conditions. The N. dimidiatum strain LJ02 mycelia cultured in potato dextrose liquid medium（PDW）were taken as the control group, and the mycelia cultured in LB medium, and the mycelia cultured in PDW medium supplemented with hydrogen peroxide, pyrazolyl ester and difenoconazole, respectively, were taken as the treatment groups. The expression stabilities of 18 candidate reference genes（CYT1, SDH2_1, TBCC, SUI1, RPL19, PPH, ATP5B, UBE2_16, RPL13, UBE2_2, PRS17, SUCLA2, ATP5A, TUB1_2, ACT1, EFTU, EF1A and GAPDH）were evaluated by real-time quantitative PCR（RT-qPCR）and stability evaluation software（geNorm, NormFinder, Bestkeeper and RefFinder）. Based on the results of RT-qPCR and software analysis, it was found that the Ct values of 18 candidate genes were within 14.80-24.66 under different culture conditions, with moderate expression levels. The reference gene with the highest stability was CYT1, followed by SUI1, while GAPDH and ACT1 were with the worst expression stability. Using at least two reference genes may improve analysis accuracy of RT-qPCR. At this time, SUCLA2 and ATP5A is the most stable reference gene combination. The results provide a theoretical basis for gene expression analysis in the biological process of N. dimidiatum.

Key words: Neoscytalidium dimidiatum, reference gene, real-time quantitative PCR, mycelial growth, expression analysis, abiotic stres, reference gene analysis software

YAO Zi-ting, CAO Xue-ying, XIAO Xue, LI Rui-fang, WEI Xiao-mei, ZOU Cheng-wu, ZHU Gui-ning. Screening of Reference Genes for RT-qPCR in Neoscytalidium dimidiatum[J]. Biotechnology Bulletin, 2023, 39(5): 92-102.

Figures/Tables 11

References 25

[1]	吕梦娇, 战春君, 白仲虎, 等. 巴斯德毕赤酵母不同生长阶段内参基因的筛选与验证[J]. 微生物学通报, 2022, 49(11): 4586-4597.
	Lü MJ, Zhan CJ, Bai ZH, et al. Identification and validation of reference genes for RT-qPCR normalization in Komagataella phaffii at different growth stages[J]. Microbiol China, 2022, 49(11): 4586-4597.
[2]	Borkowska M, Białas W, Celińska E. A new set of reference genes for comparative gene expression analyses in Yarrowia lipolytica[J]. FEMS Yeast Res, 2020, 20(7): foaa059. doi: 10.1093/femsyr/foaa059 URL
[3]	Xu ZC, Xu J, Ji AJ, et al. Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum[J]. Gene, 2015, 574(2): 352-358. doi: 10.1016/j.gene.2015.08.025 URL
[4]	Ratanaprom S, Nakkanong K, Nualsri C, et al. Overcoming encouragement of dragon fruit plant(Hylocereus undatus)against stem brown spot disease caused by Neoscytalidium dimidiatum using Bacillus subtilis combined with sodium bicarbonate[J]. Plant Pathol J, 2021, 37(3): 205-214. doi: 10.5423/PPJ.OA.01.2021.0007 pmid: 34111911
[5]	Dy KS, Wonglom P, Pornsuriya C, et al. Morphological, molecular identification and pathogenicity of Neoscytalidium dimidiatum causing stem canker of Hylocereus polyrhizus in southern Thailand[J]. Plants(Basel), 2022, 11(4): 504.
[6]	田婷婷. 马铃薯晚疫病病原菌对H₂O₂胁迫响应的机制研究[D]. 重庆: 重庆大学, 2020.
	Tian TT. The response mechanism of potato late blight pathogen to H₂O₂ stress[D]. Chongqing: Chongqing University, 2020.
[7]	齐兴柱, 刘磊, 汪军. RNA-Seq揭示Foc4在外源氧化胁迫(H₂O₂)下的基因表达及细胞代谢变化[J]. 微生物学报, 2019, 59(5): 891-906.
	Qi XZ, Liu L, Wang J. RNA-Seq reveals changes of gene expression and cellular metabolism caused by exogenous oxidative stress(H₂O₂)in Foc4[J]. Acta Microbiol Sin, 2019, 59(5): 891-906.
[8]	Camejo D, Guzmán-Cedeño Á, Moreno A. Reactive oxygen species, essential molecules, during plant-pathogen interactions[J]. Plant Physiol Biochem, 2016, 103: 10-23. doi: 10.1016/j.plaphy.2016.02.035 URL
[9]	贤小勇, 林珊宇, 朱桂宁, 等. 杀菌剂对火龙果溃疡病的室内毒力和田间防效[J]. 南方农业学报, 2018, 49(7): 1338-1345.
	Xian XY, Lin SY, Zhu GN, et al. Indoor virulence and field effects of fungicides on pitaya canker[J]. J South Agric, 2018, 49(7): 1338-1345.
[10]	蓝国兵, 何自福, 于琳, 等. 16种杀菌剂对火龙果褐腐病菌抑菌持效期及田间防效试验[J]. 广东农业科学, 2019, 46(12): 95-101.
	Lan GB, He ZF, Yu L, et al. Effective inhibition duration and field control effects of 16 fungicides against Neoscytalidium dimidiatum[J]. Guangdong Agric Sci, 2019, 46(12): 95-101.
[11]	Wan HJ, Zhao ZG, Qian CT, et al. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber[J]. Anal Biochem, 2010, 399(2): 257-261. doi: 10.1016/j.ab.2009.12.008 pmid: 20005862
[12]	Lyu XL, Shen CC, Fu YP, et al. A small secreted virulence-related protein is essential for the necrotrophic interactions of Sclerotinia sclerotiorum with its host plants[J]. PLoS Pathog, 2016, 12(2): e1005435. doi: 10.1371/journal.ppat.1005435 URL
[13]	Grube S, Göttig T, Freitag D, et al. Selection of suitable reference genes for expression analysis in human glioma using RT-qPCR[J]. J Neurooncol, 2015, 123(1): 35-42. doi: 10.1007/s11060-015-1772-7 URL
[14]	He YJ, Kim SB, Balint-Kurti P. A maize cytochrome b-c1 complex subunit protein ZmQCR7 controls variation in the hypersensitive response[J]. Planta, 2019, 249(5): 1477-1485. doi: 10.1007/s00425-019-03092-8 pmid: 30694389
[15]	Vieira A, Talhinhas P, Loureiro A, et al. Validation of RT-qPCR reference genes for in planta expression studies in Hemileia vastatrix, the causal agent of coffee leaf rust[J]. Fungal Biol, 2011, 115(9): 891-901. doi: 10.1016/j.funbio.2011.07.002 URL
[16]	Phule AS, Barbadikar KM, Madhav MS, et al. Genes encoding membrane proteins showed stable expression in rice under aerobic condition: novel set of reference genes for expression studies[J]. 3 Biotech, 2018, 8(9): 383. doi: 10.1007/s13205-018-1406-9 pmid: 30148033
[17]	周琳琳, 赵玉, 李夏雨, 等. 蚁巢伞属真菌Termitomyces clypeatus实时荧光定量PCR内参基因的筛选[J]. 菌物学报, 2022, 41(10): 1597-1606.
	Zhou LL, Zhao Y, Li XY, et al. Screening of the reference genes for RT-qPCR analysis of gene expressions in Termitomyces clypeatus[J]. Mycosystema, 2022, 41(10): 1597-1606.
[18]	盘林秀, 王娜, 王爱军, 等. 稻粒黑粉病菌实时荧光定量PCR内参基因筛选[J]. 植物病理学报, 2018, 48(5): 640-647.
	Pan LX, Wang N, Wang AJ, et al. Selection of reference genes for quantitative real-time PCR in Tilletia horrida[J]. Acta Phytopathol Sin, 2018, 48(5): 640-647.
[19]	李兵, 刘柳, 单婷婷, 等. 蜜环菌(Armillaria mellea)内参基因的筛选[J]. 微生物学通报, 2022, 49(2): 473-482.
	Li B, Liu L, Shan TT, et al. Selection of reference genes for real-time quantitative PCR of Armillaria mellea[J]. Microbiol China, 2022, 49(2): 473-482.
[20]	Chen XZ, Chen XG, Tan Q, et al. Selection of potential reference genes for RT-qPCR in the plant pathogenic fungus Colletotrichum fructicola[J]. Front Microbiol, 2022, 13: 982748. doi: 10.3389/fmicb.2022.982748 URL
[21]	苏强军, 夏樱霞, 谢放, 等. 冬虫夏草菌实时荧光定量PCR内参基因的筛选[J]. 菌物学报, 2021, 40(7): 1712-1722.
	Su QJ, Xia YX, Xie F, et al. Screening of the reference genes for qRT-PCR analysis of gene expression in Ophiocordyceps sinensis[J]. Mycosystema, 2021, 40(7): 1712-1722.
[22]	Glare EM, Divjak M, Bailey MJ, et al. Beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels[J]. Thorax, 2002, 57(9): 765-770. doi: 10.1136/thorax.57.9.765 pmid: 12200519
[23]	Tao YX, van Peer AF, Huang QH, et al. Identification of novel and robust internal control genes from Volvariella volvacea that are suitable for RT-qPCR in filamentous fungi[J]. Sci Rep, 2016, 6: 29236. doi: 10.1038/srep29236
[24]	Lin JT, Redies C. Histological evidence: housekeeping genes beta-actin and GAPDH are of limited value for normalization of gene expression[J]. Dev Genes Evol, 2012, 222(6): 369-376. doi: 10.1007/s00427-012-0420-x URL
[25]	Yang YY, Xu XY, Jing ZH, et al. Genome-wide screening and stability verification of the robust internal control genes for RT-qPCR in filamentous fungi[J]. J Fungi(Basel), 2022, 8(9): 952.

序号 No.	基因ID Gene ID	引物 Primer	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	产物长度 PCR product length/bp	基因名称 Gene name	基因注释 Gene description
Gene1	NEO_001463	gene1-qF	GCGGTTTCCAGGTCTATCGTGAAG	59	139	CYT1	Cytochrome c1
		gene1-qR	GGCTCGGTGTCGTACTCGTTCT				Cytochrome c1
Gene2	NEO_001659	gene2-qF	CGTACTGGTGGAACTCGGAGGA	58.5	151	SDH2_1	Succinate dehydrogenase subunit B
		gene2-qR	TTGAGAATGGTGTGGCAACGGTAA				Succinate dehydrogenase subunit B
Gene3	NEO_002342	gene3-qF	GCTCGCCAATCTCCGTCGTT	61.1	176	TBCC	Tubulin-specific chaperone c
		gene3-qR	TCACCACCAGCACGCTATCCT				Tubulin-specific chaperone c
Gene4	NEO_002585	gene4-qF	AGACACAGGCGAGACCAAGCA	55.8	151	SUI1	Translation initiation factor SUI1
		gene4-qR	GCAGGCAAACTTCTTCTTGATGACC				Translation initiation factor SUI1
Gene5	NEO_003941	gene5-qF	ATGCCGAGCCAGAAGTCTTTCC	57.9	117	RPL39	Ribosomal L39 protein
		gene5-qR	GGCGTTGTACCTGATGGTGTTG				Ribosomal L39 protein
Gene6	NEO_004521	gene6-qF	AAGTCTATGGCTTCTACGACGAGTG	58.4	198	PPH	Serine threonine-protein phosphatase pp2a catalytic subunit
		gene6-qR	GCGGCACCTCTTGTATCCTGTC				Serine threonine-protein phosphatase pp2a catalytic subunit
Gene7	NEO_004849	gene7-qF	GCCACCAAGTTCGCTCCTATCC	58.4	147	ATP5B	ATP synthase F1 beta subunit
		gene7-qR	GGCACCACCGAAGAGACCAATC				ATP synthase F1 beta subunit
Gene8	NEO_006464	gene8-qF	CGGTGTCTTCTTCCTCGCTATCC	58.3	183	UBE2_16	Ubiquitin-conjugating enzyme E2-16 kD
		gene8-qR	GAGCAGATGGAGAGCAGAACCTTC				Ubiquitin-conjugating enzyme E2-16 kD
Gene9	NEO_008113	gene9-qF	TCACGAGTTCGGCTGGAAGTACC	59.7	165	RPL13	Ribosomal L13 protein
		gene9-qR	TCGGCAAGCTGAGTCTTGACCTT				Ribosomal L13 protein
Gene10	NEO_008284	gene10-qF	TCTGCTCAACGACCCGAACAC	56.4	115	UBE2_2	Ubiquitin-conjugating enzyme e2_2
		gene10-qR	CCAACTCTTCTCCACCGTCTCC				Ubiquitin-conjugating enzyme e2_2
Gene11	NEO_008330	gene11-qF	CTGTCAAGAAGTCCGCCAAGGT	56.5	157	RPS17	Ribosomal protein S17e
		gene11-qR	CGCTTCATCAAGTGAGTGGTGTAG				Ribosomal protein S17e
Gene12	NEO_008873	gene12-qF	TGGTGACATTGGCTGCCTGGT	60.4	186	SUCLA2	Succinyl-CoA synthetase beta subunit
		gene12-qR	ATGTTGACGAAGATGGCGGTGAC				Succinyl-CoA synthetase beta subunit
Gene13	NEO_010290	gene13-qF	TCCTCTCATCTTCGCTGGTGTCA	56.2	110	ATP5A	ATP synthase F1 alpha subunit
		gene13-qR	GCTCGGACTCGTTGCTCTTCAG				ATP synthase F1 alpha subunit
Gene14	NEO_010575	gene14-qF	CAAGGAGGATGCCGCCAACAA	59	145	TUB1_2	Tubulin alpha-2 chain
		gene14-qR	ACCGCCGAAGGAGTGGAAGAT				Tubulin alpha-2 chain
Gene15	NEO_011231	gene15-qF	CCAAGTCCAACCGTGAGAAGATGA	56.7	102	ACT1	Actin
		gene15-qR	CGGAAGCGTACAGCGACAGAA				Actin
Gene16	NEO_011865	gene16-qF	AGACCGACAACCGCCACTATGC	60.1	184	EFTU	Translation elongation factor Tu
		gene16-qR	ACAATCTTCTGGACACCGACCTGAC				Translation elongation factor Tu
Gene17	NEO_012815	gene17-qF	CTGGTGAGTTCGAGGCTGGTATCT	58.2	121	EF1A	Translation elongation factor 1-alpha protein
		gene17-qR	CACTTGGTGGTGTCCATCTTGTTGA				Translation elongation factor 1-alpha protein
Gene18	NEO_013111	gene18-qF	CCGTACCGCTGCTACCAACATC	59.8	195	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase
		gene18-qR	GCCTTGATAGTCGCCTTGATCTCAT				Glyceraldehyde-3-phosphate dehydrogenase

序号 No.	基因ID Gene ID	引物 Primer	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃	产物长度 PCR product length/bp	基因名称 Gene name	基因注释 Gene description
Gene1	NEO_001463	gene1-qF	GCGGTTTCCAGGTCTATCGTGAAG	59	139	CYT1	Cytochrome c1
		gene1-qR	GGCTCGGTGTCGTACTCGTTCT				Cytochrome c1
Gene2	NEO_001659	gene2-qF	CGTACTGGTGGAACTCGGAGGA	58.5	151	SDH2_1	Succinate dehydrogenase subunit B
		gene2-qR	TTGAGAATGGTGTGGCAACGGTAA				Succinate dehydrogenase subunit B
Gene3	NEO_002342	gene3-qF	GCTCGCCAATCTCCGTCGTT	61.1	176	TBCC	Tubulin-specific chaperone c
		gene3-qR	TCACCACCAGCACGCTATCCT				Tubulin-specific chaperone c
Gene4	NEO_002585	gene4-qF	AGACACAGGCGAGACCAAGCA	55.8	151	SUI1	Translation initiation factor SUI1
		gene4-qR	GCAGGCAAACTTCTTCTTGATGACC				Translation initiation factor SUI1
Gene5	NEO_003941	gene5-qF	ATGCCGAGCCAGAAGTCTTTCC	57.9	117	RPL39	Ribosomal L39 protein
		gene5-qR	GGCGTTGTACCTGATGGTGTTG				Ribosomal L39 protein
Gene6	NEO_004521	gene6-qF	AAGTCTATGGCTTCTACGACGAGTG	58.4	198	PPH	Serine threonine-protein phosphatase pp2a catalytic subunit
		gene6-qR	GCGGCACCTCTTGTATCCTGTC				Serine threonine-protein phosphatase pp2a catalytic subunit
Gene7	NEO_004849	gene7-qF	GCCACCAAGTTCGCTCCTATCC	58.4	147	ATP5B	ATP synthase F1 beta subunit
		gene7-qR	GGCACCACCGAAGAGACCAATC				ATP synthase F1 beta subunit
Gene8	NEO_006464	gene8-qF	CGGTGTCTTCTTCCTCGCTATCC	58.3	183	UBE2_16	Ubiquitin-conjugating enzyme E2-16 kD
		gene8-qR	GAGCAGATGGAGAGCAGAACCTTC				Ubiquitin-conjugating enzyme E2-16 kD
Gene9	NEO_008113	gene9-qF	TCACGAGTTCGGCTGGAAGTACC	59.7	165	RPL13	Ribosomal L13 protein
		gene9-qR	TCGGCAAGCTGAGTCTTGACCTT				Ribosomal L13 protein
Gene10	NEO_008284	gene10-qF	TCTGCTCAACGACCCGAACAC	56.4	115	UBE2_2	Ubiquitin-conjugating enzyme e2_2
		gene10-qR	CCAACTCTTCTCCACCGTCTCC				Ubiquitin-conjugating enzyme e2_2
Gene11	NEO_008330	gene11-qF	CTGTCAAGAAGTCCGCCAAGGT	56.5	157	RPS17	Ribosomal protein S17e
		gene11-qR	CGCTTCATCAAGTGAGTGGTGTAG				Ribosomal protein S17e
Gene12	NEO_008873	gene12-qF	TGGTGACATTGGCTGCCTGGT	60.4	186	SUCLA2	Succinyl-CoA synthetase beta subunit
		gene12-qR	ATGTTGACGAAGATGGCGGTGAC				Succinyl-CoA synthetase beta subunit
Gene13	NEO_010290	gene13-qF	TCCTCTCATCTTCGCTGGTGTCA	56.2	110	ATP5A	ATP synthase F1 alpha subunit
		gene13-qR	GCTCGGACTCGTTGCTCTTCAG				ATP synthase F1 alpha subunit
Gene14	NEO_010575	gene14-qF	CAAGGAGGATGCCGCCAACAA	59	145	TUB1_2	Tubulin alpha-2 chain
		gene14-qR	ACCGCCGAAGGAGTGGAAGAT				Tubulin alpha-2 chain
Gene15	NEO_011231	gene15-qF	CCAAGTCCAACCGTGAGAAGATGA	56.7	102	ACT1	Actin
		gene15-qR	CGGAAGCGTACAGCGACAGAA				Actin
Gene16	NEO_011865	gene16-qF	AGACCGACAACCGCCACTATGC	60.1	184	EFTU	Translation elongation factor Tu
		gene16-qR	ACAATCTTCTGGACACCGACCTGAC				Translation elongation factor Tu
Gene17	NEO_012815	gene17-qF	CTGGTGAGTTCGAGGCTGGTATCT	58.2	121	EF1A	Translation elongation factor 1-alpha protein
		gene17-qR	CACTTGGTGGTGTCCATCTTGTTGA				Translation elongation factor 1-alpha protein
Gene18	NEO_013111	gene18-qF	CCGTACCGCTGCTACCAACATC	59.8	195	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase
		gene18-qR	GCCTTGATAGTCGCCTTGATCTCAT				Glyceraldehyde-3-phosphate dehydrogenase

序号 No.	内参基因 Reference gene	扩增效率 RT-qPCR efficiency/%	相关系数 Correlation coefficient R²	序号 No.	内参基因 Reference gene	扩增效率 RT-qPCR efficiency/%	相关系数 Correlation coefficient R²
Gene 1	CYT1	106.4	0.998 5	Gene 10	UBE2_2	96.5	0.999 9
Gene 2	SDH2_1	106.2	0.986 5	Gene 11	RPS17	96.7	0.996 6
Gene 3	TBCC	104.3	0.989 4	Gene 12	SUCLA2	103.6	0.996 6
Gene 4	SUI1	100.1	0.999 5	Gene 13	ATP5A	101.9	0.996 9
Gene 5	RPL39	108.3	0.998 8	Gene 14	TUB1_2	93.6	0.999 6
Gene 6	PPH	108.9	0.999 8	Gene 15	ACT1	91.6	0.999 9
Gene 7	ATP5B	104.7	0.997 4	Gene 16	EFTU	102.4	0.994 1
Gene 8	UBE2_16	104.2	0.999 9	Gene 17	EF1A	94.8	0.996 8
Gene 9	RPL13	100.9	0.999 9	Gene 18	GAPDH	90.9	0.995 0

序号 No.	内参基因 Reference gene	扩增效率 RT-qPCR efficiency/%	相关系数 Correlation coefficient R²	序号 No.	内参基因 Reference gene	扩增效率 RT-qPCR efficiency/%	相关系数 Correlation coefficient R²
Gene 1	CYT1	106.4	0.998 5	Gene 10	UBE2_2	96.5	0.999 9
Gene 2	SDH2_1	106.2	0.986 5	Gene 11	RPS17	96.7	0.996 6
Gene 3	TBCC	104.3	0.989 4	Gene 12	SUCLA2	103.6	0.996 6
Gene 4	SUI1	100.1	0.999 5	Gene 13	ATP5A	101.9	0.996 9
Gene 5	RPL39	108.3	0.998 8	Gene 14	TUB1_2	93.6	0.999 6
Gene 6	PPH	108.9	0.999 8	Gene 15	ACT1	91.6	0.999 9
Gene 7	ATP5B	104.7	0.997 4	Gene 16	EFTU	102.4	0.994 1
Gene 8	UBE2_16	104.2	0.999 9	Gene 17	EF1A	94.8	0.996 8
Gene 9	RPL13	100.9	0.999 9	Gene 18	GAPDH	90.9	0.995 0

基因序号 No.	基因名称 Gene name	几何平均值 Geo mean	算术平均值 AR mean	最小值 Min	最大值 Max	相关系数 Correlation coefficient（r）	标准偏差 Std dev	变异系数 CV
Gene1	CYT1	19.94	19.94	19.24	20.68	0.960	0.31	1.53
Gene2	SDH2_1	19.69	19.69	19.06	20.16	0.754	0.30	1.54
Gene3	TBCC	22.90	22.92	21.50	24.66	0.915	0.83	3.63
Gene4	SUI1	17.65	17.66	16.58	18.49	0.952	0.47	2.66
Gene5	RPL39	19.71	19.72	18.77	20.67	0.851	0.63	3.21
Gene6	PPH	21.46	21.47	20.20	22.48	0.936	0.72	3.38
Gene7	ATP5B	18.21	18.22	17.97	18.74	0.521	0.20	1.11
Gene8	UBE2_16	18.16	18.17	17.48	19.18	0.912	0.44	2.40
Gene9	RPL13	19.97	19.98	18.81	20.69	0.761	0.56	2.79
Gene10	UBE2_2	20.47	20.49	19.63	21.76	0.963	0.53	2.6
Gene11	RPS17	19.82	19.83	18.73	20.78	0.803	0.61	3.09
Gene12	SUCLA2	20.78	20.79	20.15	21.48	0.825	0.38	1.81
Gene13	ATP5A	19.49	19.50	18.88	20.03	0.87	0.33	1.67
Gene14	TUB1_2	22.06	22.07	21.31	23.66	0.892	0.55	2.50
Gene15	ACT1	21.30	21.33	19.76	22.72	0.835	1.13	5.29
Gene16	EFTU	21.48	21.49	20.66	22.36	0.577	0.65	3.00
Gene17	EF1A	16.04	16.05	15.35	16.48	0.893	0.31	1.96
Gene18	GAPDH	15.97	16.00	14.80	17.62	0.929	0.89	5.56