刺槐实时定量PCR分析中内参基因的选择

doi:10.11707/j.1001-7488.20140923

Abstract

Abstract:

The real-time PCR is an important method for analyzing gene expression levels. Selection of reference genes suitable for calibration of the expression of objective gene according to specific experimental materials and conditions is a prerequisite for obtaining reliable RT-PCR results. In this study, we used different individuals and different organs of black locust(Robinia pseudoacacia)to test the stability of six internal genes such as Actin gene (ACT), 18S ribosomal RNA ( 18S rRNA), adenosylmethionine decarboxylase gene (SAMDC), helicase gene (Helicase), elongation factor gene ( EF1 -α), glyceraldehyde-3-phosphate-dehydrogenase gene (GAPDH) in the real-time PCR. After being analyzed with Genorm and NormFinder, the results showed that the ACT and 18S rRNA gene were the most stable genes by using different plant individuals as experimental material, while the ACT and GAPDH were the most stable genes with using different tissues and organs. Since some controversies existed with the 18S rRNA as reference gene, the both ACT and GAPDH used for reference gene in various test conditions obtained more accurate results. This study has important implications in obtaining a more accurate result of quantitative expression of black locust.

Key words: Robinia pseudoacacia, reference gene, real-time quantitative PCR, GeNorm, NormFinder

CLC Number:

S718.46

Wang Jinxing, Zhang Lijun, Liao Ziyi, Zhang Yungen, Qiu Qiandong, Sun Peng, Sun Yuhan, Hu Ruiyang, Lu Nan, Li Yun. The Selection of Reference Genes for Real-Time Quantitative PCR Normalization in Black Locust(Robinia pseudoacacia)[J]. Scientia Silvae Sinicae, 2014, 50(9): 167-172.

References

孙鹏, 戴丽, 胡瑞阳, 等. 2012. 刺槐开花传粉及交配方式. 东北林业大学学报, 40(1): 6-12.
(Sun P, Dai L, Hu R Y, et al. 2012. Flowering characteristics and pollination and mating patterns of Robinia pseudoacacia. Journal of Northeast Forestry University, 40(1): 6-12. [in Chinese] )
王彦杰, 董丽, 张超, 等. 2012. 牡丹实时定量PCR分析中内参基因的选择. 农业生物技术学报, 20(5): 521-528.
(Wang Y J, Dong L, Zhang C, et al. 2012. Reference gene selection for real-time quantitative PCR normalization in tree peony (Paeonia suffruticosa Andr.). Journal of Agricultural Biotechnology, 20(5): 521-528. [in Chinese] )
魏永赞, 赖彪, 胡福初, 等. 2012. 用于荔枝qPCR分析的内参基因克隆及稳定性分析. 华南农业大学学报, 33(3):301-306.
(Wei Y Z, Lai B, Hu F C, et al. 2012. Cloning and stability analysis of reference genes for expression studies by quantitative real-time PCR in Litchi. Journal of South China Agricultural University, 33(3):301-306. [in Chinese] )
习洋, 胡瑞阳, 王欢, 等. 2012. 刺槐未成熟合子胚的体细胞胚胎发生和植株再生. 林业科学, 48(1): 60-69.
(Xi Y, Hu R Y, Wang H, et al. 2012. Somatic embryogenesis and plant regeneration from immature zygotic embryos of black locust (Robinia pseudoacacia). Scientia Silvae Sinicae, 48(1): 60-69. [in Chinese] )
袁伟, 万红建, 杨悦俭. 2012. 植物实时荧光定量PCR内参基因的特点及选择. 植物学报, 47(4): 427-436.
(Yuan W, Wan H J, Yang Y J. 2012. Characterization and selection of reference genes for real-time quantitative RT-PCR of plants. Bulletin of Botany, 47(4): 427-436. [in Chinese] )
袁亚男, 刘文忠. 2008. 实时荧光定量PCR技术的类型、特点与应用. 中国畜牧兽医, 35(3): 72.
(Yuan Y N, Liu W Z. 2008. The types,characteristics and applications of real-time fluoescent quantitative PCR techniques. China Animal Husbandry & Veterinary Medicine, 35(3): 72.
赵文静, 徐洁, 包秋华,等. 2010. 实时荧光定量PCR中内参基因的选择. 微生物通报, 37(12): 1825-1829.
(Zhao W J, Xu J, Bao Q H, et al. 2010. Selection of reference genes for real-time quantitative PCR. Microbiology China, 37(12): 1825-1829. [in Chinese] )
Bustin S A, Beaulieu J F, Huggett J, et al. 2009. The MIQE guideline: Minemum information for publication of quantitative real-time PCR experience. Clinical Chemistry, 55(4): 611-622.
Chang Ermei, Shi Shengqing, Liu Jianfeng, et al. 2012. Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) using real-time PCR. PLOS ONE, 7(3): e33278.
Giulietti A, Overbergh L, Valckx D, et al. 2001. An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods, 25(4): 386-401.
Gutierrez L, Mauriat M, Guenin S, et al. 2008. The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J, 6(6): 609-618.
Hellemans J, Mortier G, Paepe D A, et al. 2007. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biology, 8(2): R19.
Hu R, Fan C M, Li H Y, et al. 2009. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol, 10: 93.
Huggett J, Dheda K, Bustin S, et al. 2005. Real-time RT-PCR normalization: strategies and considerations. Genes Immun, 6(6): 279-284.
Iskandar H M, Simpeon R S, Casu R E, et al. 2004. Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Mol Biol Rep, 22(4): 325-337.
Jain M. 2009. Genome-wide identification of novel internal control genes for normalization of gene expression during various stages of development in rice. Plant Sci, 176(5): 702-706.
Jian B, Liu B, Bi Y R, et al. 2008. Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol, 9: 59.
Keeler H L. 1990. Our native trees and how to identify them. New York: Charles Scriber’s Sons, 97-102.
Long Xiangyu, Wang Jirui, Ouellet T, et al. 2010. Genome wide identification and evaluation of novel internal control genes for qPCR based transcript normalization in wheat. Plant Molecular Biology, 74(3): 307-311.
Mackay I M. 2004. Real-time PCR in the microbiology laboratory. Clin Microbiol Infect, 10(3):190-212.
Mascia T, Santovito E, Gallitelli D, et al. 2010. Evalution of reference genes for quantitative reverse transcription polymerase chain reaction normalization in infected tomato plants. Mol Plant Pathol, 11(6): 805-816.
Nolan T, Hands R E, Bustin S A. 2006. Quantification of mRNA using real-time RT-PCR. Nat Protoc, 1(3):1559-1582.
Paolacci A R, Tanzarella O A, Porceddu E, et al. 2009. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol, 10: 11.
Pfaffl M W, Horgan G W, Dempfle L. 2002. Relative Expression Software Tool for group wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res, 30(9):e36.
Pfaffl M W, Tichopad A, Prgomet C, et al. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper-excel-based tool using pair-wise correlations. Biotechnol Lett, 26(6): 509-515.
Postollec F, Falentin H, Pavan S, et al. 2011. Recent advances in quantitative PCR aplications in food microbiology. Food Microbiology, 28(5): 848-861.
Remans T, Smeets K, Opdenakker K, et al. 2008. Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta, 227(6): 1343-1349.
Schmid H, Cohen C D, Henger A, et al. 2003. Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies. Kidney Int, 64: 356-360.
Stürzenbaum S R, Kille P. 2001. Control genes in quantitative molecular biological techniques: The variability of invariance. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 130(3): 281-289.
Vandesompele J, De Preter K, Pattyn F, et al. 2002. Accurate normalization of real-time quantitative RT-PCR data by genometric averaging of multiple internal control genes. Genome Biology, 3(7): research0034:1-11.

[1]	Zhang Xiaoling, Wang Liuqiang, Sun Pei, Wu Lishuan, Fan Wei, Zhang Jin, Lu Mengzhu, Hu Jianjun. Differences in Pollen Germination and Allergenic Proteins among Different Populus Germplasms [J]. Scientia Silvae Sinicae, 2017, 53(2): 54-64.
[2]	Chu Wenyuan, Wang Yujiao, Zhu Dongyue, Chen Zhu, Yan Hanwei, Xiang Yan. Selection of Novel Reference Genes in Poplar under Salt and Drought Stresses [J]. Scientia Silvae Sinicae, 2017, 53(10): 70-79.
[3]	Liu Wenzhe, Niu Mingyue, Li Xiuyun, Lin Erpei, Huang Huahong, Tong Zaikang. The Selection of Reference Genes for Quantitative PCR in Betula luminifera [J]. Scientia Silvae Sinicae, 2016, 52(8): 29-37.
[4]	Cheng Longping, Hu Haitao, Guo Weidong, Yang Li, Wang Changchun, Yang Ling. Evaluation and Validation of Potential Reference Genes for Quantitative Real-Time PCR Analysis in Elaeagnus umbellata [J]. Scientia Silvae Sinicae, 2015, 51(5): 135-144.
[5]	Duan Xuwei;Deng Shu;Shen Yuanyue;Feng Yongqing;Qin Ling. Cloning and Expression Analysis of CmAPs in Mutant Short Catkin of Castanea mollissima [J]. Scientia Silvae Sinicae, 2010, 46(12): 49-55.
[6]	Wen Qiaofu;Shen Hongxiang;Yao Yuncong;Tian Ji;Song Tingting. Cloning and Expression of McDFR gene in the Different Foliar Color Cultivars of Maluscrabapple [J]. Scientia Silvae Sinicae, 2010, 46(11): 16-24.

The Selection of Reference Genes for Real-Time Quantitative PCR Normalization in Black Locust(Robinia pseudoacacia)

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 6

Recommended Articles

Metrics

Comments