Screening of Reference Genes for Quantitative Real-time PCR Analysis in Asarum sieboldii

doi:10.13560/j.cnki.biotech.bull.1985.2017-0352

Abstract

Abstract: Selecting appropriate reference gene is key step for the accurate analysis of the expression variations of target genes. Using several common-used reference genes of SAND-1,ACT,18S rRNA,CYP2,GAPDH and TUB as candidate genes and quantitative real-time PCR analysis of gene expression,the expression stabilities of these 6 candidate genes in various samples from roots,rhizomes,leaves,petioles and flowers in different growth stages of nutrition growth,flowering and post-flowering were assessed,with a set of approaches such as geNorm,NormFinder,BestKeeper and Delta CT,representatively. It is for screening the appropriate reference genes in analysing gene expressions of Asarum sieboldiiMiq. in varied tissues at different growth stage. The results demonstrated that 18S rRNA was the optimal object to act as a proper reference gene in this species because it displayed the most stable expression in almost all samples. This study will provide a feasible and valid calibrate not only for gene function characterization concerning chemical compound biosynthesis but also for gene differential expression analysis in A. sieboldii,ensuring the accuracy and reliability of results.

Key words: Asarum sieboldii, reference gene, GeNorm, NormFinder, BestKeeper, Delta CT

ZHAO Xiao-bing, PAN Lei, LIU Wei, LIN Mao-yi, LI Hong-qing, LIU Zhong. Screening of Reference Genes for Quantitative Real-time PCR Analysis in Asarum sieboldii[J]. Biotechnology Bulletin, 2017, 33(11): 174-179.

References

[1] 国家药典委员会. 《中华人民共和国药典》（2015版一部）[M]. 北京：中国医药科技出版社, 2015.
[2] 王晓丽, 金礼吉, 续繁星, 等. 中草药细辛研究进展[J]. 亚太传统医药, 2013（97）：68-71.
[3] 韩俊艳, 孙川力, 纪明山. 中药细辛的研究进展[J]. 中国农学通报, 2011, 27（9）：46-50.
[4] Valasek MA, Repa JJ. The power of real-time PCR[J]. Advances in Physiology Education, 2005, 29（3）：151-159.
[5] Mackay IM. Real-time PCR in the microbiology laboratory[J]. Clinical Microbiology and Infection, 2004, 10（3）：190-212.
[6] 赵焕英, 包金凤. 实时荧光定量PCR技术的原理及其应用研究进展[J]. 中国组织化学和细胞化学杂志, 2007, 16（4）：492-497.
[7] Andersen CL, Jensen JL, Ørntoft TF. 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 Research, 2004, 64（15）：5245-5250.
[8] 张艳君, 朱志峰, 陆融. 基因表达转录分析中内参基因的选择[J]. 生物化学与生物物理进展, 2007, 34（5）：546-550.
[9] Guénin S, Mauriat M, Pelloux J, et al. Normalization of qRT-PCR data：the necessity of adopting a systematic, experimental conditions-specific, validation of references[J]. Journal of Experimental Botany, 2009, 60（2）：487-493.
[10] 周兰, 张利义, 张彩霞, 等. 苹果实时荧光定量PCR分析中内参基因的筛选[J]. 果树学报, 2012（6）：965-970.
[11] 吴文凯, 刘成前, 周志刚, 等. 用于莱茵衣藻荧光定量PCR分析的内参基因选择[J]. 植物生理学报, 2009, 45（7）：667-672.
[12] 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 Biology, 2002, 3（7）：research0034. 1.
[13] Pfaffl MW, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity：BestKeeper-Excel-based tool using pair-wise correlations[J]. Biotechnology Letters, 2004, 26（6）：509-515.
[14] Silver N, Best S, Jiang J, et al. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR[J]. BMC Molecular Biology, 2006, 7（1）：33.
[15] Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR[J]. Nature Protocols, 2006, 1（3）：1559-1582.
[16] Dheda K, Huggett JF, Chang JS, et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization[J]. Analytical Biochemistry, 2005, 344（1）：141-143.
[17] Dheda K, Huggett JF, Bustin SA, et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR[J]. Biotechniques, 2004, 37：112-119.
[18] 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, 45：579-587.
[19] Iskandar HM, Simpson RS, Casu RE, et al. Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane[J]. Plant Molecular Biology Reporter, 2004, 22（4）：325-337.
[20] 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]. Molecular Plant Pathology, 2010, 11（6）：805-816.