Stability Analysis of Reference Genes for Larch Gene Expression Studies by Quantitative Real-Time PCR

WU Tao; LI Wan-feng; ZHANG Jun-hong; HAN Su-ying; YANG Wen-hua; QI Li-wang

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Stability Analysis of Reference Genes for Larch Gene Expression Studies by Quantitative Real-Time PCR

1.
Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
2.
Yunnan Laboratory for Conservation of the Rare, Endangered and Endemic Forest Plants of State Forestry Administration, Yunnan Key Laboratory for Forest Plant Cultivation and Utilization
3.
Yunnan Academy of Forestry, Kunming 650201, Yunnan, China

Received Date: 2013-08-22

Abstract

The selection of suitable reference gene is an important prerequisite for successful gene expression analysis by quantitative real-time PCR (qPCR). The authors investigated the expression stability of 12 endogenous house-keeping genes (ACT4, APm, Chc, Gapc, RPL1, RPL2, EF1, EF2, eIF, E3UL, UBQ and UPL2) in 31 samples of Larix kaempferi (Lamb.) Carr. including needles, stems, roots, pollens, zygotic embryos and somatic embryos. The GeNorm and NormFinder algorithms analysis reveals that the APm is the most stable gene among different organs and developmental stages (somatic embryogenesis and seed germination), EF1 and eIF are most stable among stems and needles during plant growth (90 days, 1.5 years, 5 years, 10 years, 25 years and 50 year), respectively. The results would provide optimum internal reference genes for larch gene expression analysis using qPCR.
- Larix,
- house-keeping gene,
- qPCR,
- gene expression,
- stability

References

[1]	Mascia T, Santovito E, Gallitelli D, et al. Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants[J]. Mol Plant Pathol., 2010, 11: 805-816
[2]	Heid C A, Stevens J, Livak K J, et al. Real time quantitative PCR[J]. Genome Res, 1996,6 (10): 986-994
[3]	Radonic A, Thulke S, Mackay I M, et al. Guideline to reference gene selection for quantitative real-time PCR[J]. Biochemical and Biophysical Research Communications, 2004, 313: 856-862
[4]	胡瑞波, 范成明, 傅永福. 植物实时荧光定量PCR内参基因的选择[J]. 中国农业科技导报, 2009, 11(6): 30-36
[5]	Lee J M, Roche J R, Donaghy D J, et al. Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)[J]. BMC Mol Biol, 2010, 11: 8
[6]	de Oliveira L A, Breton M C, Bastolla F M, et al. Reference genes for the normalization of gene expression in Eucalyptus species[J]. Plant Cell Physiol, 2012, 53 (2): 405-422
[7]	Czechowski T,Stitt M,Ahnmnn T, et al. Genome wide identification and testing of superior reference genes for transcript normalization in Arabidopsis[J]. Plant Physio1, 2005, 139: 5-17
[8]	Brunner A M, Yakovlev I A, Strauss S H. Validating internal controls for quantitative plant gene expression studies[J]. BMC Plant Biology, 2004, 4: 14
[9]	Udvardi M K, Czechowski T, Scheible W R. Eleven golden rules of quantitative RT-PCR[J]. Plant Cell, 2008, 20(7): 1736-1737
[10]	Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 3 (7): RESEARCH0034
[11]	Andersen C L, Jensen J L, Orntoft T F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Res, 2004, 64 (15): 5245-5250
[12]	Zhang Yuan, Zhang Shougong, Han Suying, et al. Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch (Larix leptolepis)[J]. Plant Cell Reports, 2012, 31(9): 1637-1657
[13]	袁伟, 万红建, 杨悦俭. 植物实时荧光定量PCR内参基因的特点及选择[J]. 植物学报, 2012, 47(4): 427-436
[14]	Exposito-Rodriguez M, Borges A A, Borges-Perez A, et al. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process[J]. BMC Plant Biol, 2008, 8: 131
[15]	Reid K E, Olsson N, Schlosser J, et al. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development[J]. BMC Plant Biol, 2006, 6: 27
[16]	涂礼莉, 张献龙, 刘迪秋, 等. 棉花纤维发育和体细胞胚发生过程中实时定量PCR内对照基因的筛选[J]. 科学通报, 2007, 52(20): 2379-2385
[17]	孙美莲, 王云生, 杨冬青, 等. 茶树实时荧光定量PCR分析中内参基因的选择[J]. 植物学报, 2010, 45 (5): 579-587
[18]	Jian B, Liu B, Bi Y, et al. Validation of internal control for gene expression study in soybean by quantitative real-time PCR[J]. BMC Mol Biol, 2008, 9: 59
[19]	李爱, 刘超, 韩春乐, 等. 落叶松优势杂交子代与亲本间基因组DNA甲基化变异研究[J]. 南开大学学报:自然科学版, 2012, 45(5): 66-71
[20]	Zhang Shougong, Han Suying, Li Wanfeng, et al. miRNA regulation in fast and slow-growing hybrid Larix trees[J]. Trees, 2012, 26(5): 1597-1604
[21]	Zhang Junhong, Zhang Shougong, Han Suying, et al. Genome-wide identification of microRNAs in Japanese larch and stage-specific modulation of eleven conserved microRNAs and their targets during somatic embryogenesis[J]. Planta, 2012, 236(2): 647-657
[22]	Li Wanfeng, Zhang Shougong, Han Suying, et al. Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr.[J]. Plant Cell, Tissue and Organ Culture, 2013, 113(1): 131-136

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Stability Analysis of Reference Genes for Larch Gene Expression Studies by Quantitative Real-Time PCR

1. Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

2. Yunnan Laboratory for Conservation of the Rare, Endangered and Endemic Forest Plants of State Forestry Administration, Yunnan Key Laboratory for Forest Plant Cultivation and Utilization

3. Yunnan Academy of Forestry, Kunming 650201, Yunnan, China

Received Date: 2013-08-22

Abstract: The selection of suitable reference gene is an important prerequisite for successful gene expression analysis by quantitative real-time PCR (qPCR). The authors investigated the expression stability of 12 endogenous house-keeping genes (ACT4, APm, Chc, Gapc, RPL1, RPL2, EF1, EF2, eIF, E3UL, UBQ and UPL2) in 31 samples of Larix kaempferi (Lamb.) Carr. including needles, stems, roots, pollens, zygotic embryos and somatic embryos. The GeNorm and NormFinder algorithms analysis reveals that the APm is the most stable gene among different organs and developmental stages (somatic embryogenesis and seed germination), EF1 and eIF are most stable among stems and needles during plant growth (90 days, 1.5 years, 5 years, 10 years, 25 years and 50 year), respectively. The results would provide optimum internal reference genes for larch gene expression analysis using qPCR.

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Reference (22)

[1]	Mascia T, Santovito E, Gallitelli D, et al. Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants[J]. Mol Plant Pathol., 2010, 11: 805-816
[2]	Heid C A, Stevens J, Livak K J, et al. Real time quantitative PCR[J]. Genome Res, 1996,6 (10): 986-994
[3]	Radonic A, Thulke S, Mackay I M, et al. Guideline to reference gene selection for quantitative real-time PCR[J]. Biochemical and Biophysical Research Communications, 2004, 313: 856-862
[4]	胡瑞波, 范成明, 傅永福. 植物实时荧光定量PCR内参基因的选择[J]. 中国农业科技导报, 2009, 11(6): 30-36
[5]	Lee J M, Roche J R, Donaghy D J, et al. Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)[J]. BMC Mol Biol, 2010, 11: 8
[6]	de Oliveira L A, Breton M C, Bastolla F M, et al. Reference genes for the normalization of gene expression in Eucalyptus species[J]. Plant Cell Physiol, 2012, 53 (2): 405-422
[7]	Czechowski T,Stitt M,Ahnmnn T, et al. Genome wide identification and testing of superior reference genes for transcript normalization in Arabidopsis[J]. Plant Physio1, 2005, 139: 5-17
[8]	Brunner A M, Yakovlev I A, Strauss S H. Validating internal controls for quantitative plant gene expression studies[J]. BMC Plant Biology, 2004, 4: 14
[9]	Udvardi M K, Czechowski T, Scheible W R. Eleven golden rules of quantitative RT-PCR[J]. Plant Cell, 2008, 20(7): 1736-1737
[10]	Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biol, 2002, 3 (7): RESEARCH0034
[11]	Andersen C L, Jensen J L, Orntoft T F. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Res, 2004, 64 (15): 5245-5250
[12]	Zhang Yuan, Zhang Shougong, Han Suying, et al. Transcriptome profiling and in silico analysis of somatic embryos in Japanese larch (Larix leptolepis)[J]. Plant Cell Reports, 2012, 31(9): 1637-1657
[13]	袁伟, 万红建, 杨悦俭. 植物实时荧光定量PCR内参基因的特点及选择[J]. 植物学报, 2012, 47(4): 427-436
[14]	Exposito-Rodriguez M, Borges A A, Borges-Perez A, et al. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process[J]. BMC Plant Biol, 2008, 8: 131
[15]	Reid K E, Olsson N, Schlosser J, et al. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development[J]. BMC Plant Biol, 2006, 6: 27
[16]	涂礼莉, 张献龙, 刘迪秋, 等. 棉花纤维发育和体细胞胚发生过程中实时定量PCR内对照基因的筛选[J]. 科学通报, 2007, 52(20): 2379-2385
[17]	孙美莲, 王云生, 杨冬青, 等. 茶树实时荧光定量PCR分析中内参基因的选择[J]. 植物学报, 2010, 45 (5): 579-587
[18]	Jian B, Liu B, Bi Y, et al. Validation of internal control for gene expression study in soybean by quantitative real-time PCR[J]. BMC Mol Biol, 2008, 9: 59
[19]	李爱, 刘超, 韩春乐, 等. 落叶松优势杂交子代与亲本间基因组DNA甲基化变异研究[J]. 南开大学学报:自然科学版, 2012, 45(5): 66-71
[20]	Zhang Shougong, Han Suying, Li Wanfeng, et al. miRNA regulation in fast and slow-growing hybrid Larix trees[J]. Trees, 2012, 26(5): 1597-1604
[21]	Zhang Junhong, Zhang Shougong, Han Suying, et al. Genome-wide identification of microRNAs in Japanese larch and stage-specific modulation of eleven conserved microRNAs and their targets during somatic embryogenesis[J]. Planta, 2012, 236(2): 647-657
[22]	Li Wanfeng, Zhang Shougong, Han Suying, et al. Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr.[J]. Plant Cell, Tissue and Organ Culture, 2013, 113(1): 131-136

Stability Analysis of Reference Genes for Larch Gene Expression Studies by Quantitative Real-Time PCR

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通讯作者: 陈斌, bchen63@163.com

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Stability Analysis of Reference Genes for Larch Gene Expression Studies by Quantitative Real-Time PCR

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