Advertisement

Current Microbiology

, Volume 68, Issue 1, pp 120–126 | Cite as

Identification and Evaluation of Reference Genes for qRT-PCR Normalization in Ganoderma lucidum

  • Jiang Xu
  • ZhiChao Xu
  • YingJie Zhu
  • HongMei Luo
  • Jun Qian
  • AiJia Ji
  • YuanLei Hu
  • Wei Sun
  • Bo Wang
  • JingYuan Song
  • Chao Sun
  • ShiLin Chen
Article

Abstract

Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs—geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and β-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis.

Keywords

Reference Gene Fruiting Body Candidate Reference Gene Ganoderma Medicinal Mushroom 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

This work was supported by the Program for Changjiang Scholars and Innovative Research Team in University from Ministry of Education of China (Grant No. 2012IRT1150).

Supplementary material

284_2013_442_MOESM1_ESM.docx (259 kb)
Supplementary material 1 (DOCX 259 kb)

References

  1. 1.
    Andersen CL, Jensen JL, Orntoft TF (2004) 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. Cancer Res 64:5245–5250PubMedCrossRefGoogle Scholar
  2. 2.
    Boh B, Berovic M, Zhang J, Zhi-Bin L (2007) Ganoderma lucidum and its pharmaceutically active compounds. Biotechnol Annu Rev 13:265–301PubMedCrossRefGoogle Scholar
  3. 3.
    Cao S, Zhang X, Ye N, Fan X, Mou S, Xu D, Liang C, Wang Y, Wang W (2012) Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR. Biochem Biophys Res Commun 424:118–123PubMedCrossRefGoogle Scholar
  4. 4.
    Chen S, Xu J, Liu C et al (2012) Genome sequence of the model medicinal mushroom Ganoderma lucidum. Nat Commun 3:913PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Carrillo-Casas Erika Margarita, Hernández-Castro Rigoberto, Suárez-Güemes Francisco, de la Peña-Moctezuma Alejandro (2008) Selection of the internal control gene for real-time quantitative RT-PCR assays in temperature treated Leptospira. Curr Microbiol 56:539–546PubMedCrossRefGoogle Scholar
  6. 6.
    Fang QH, Zhong JJ (2002) Two-stage culture process for improved production of ganoderic acid by liquid fermentation of higher fungus Ganoderma lucidum. Biotechnol Prog 18:51–54PubMedCrossRefGoogle Scholar
  7. 7.
    Ferguson JA, Boles TC, Adams CP, Walt DR (1996) A fiber-optic DNA biosensor microarray for the analysis of gene expression. Nat Biotechnol 14:1681–1684PubMedCrossRefGoogle Scholar
  8. 8.
    Han X, Lu M, Chen Y, Zhan Z, Cui Q, Wang Y (2012) Selection of reliable reference genes for gene expression studies using real-time PCR in tung tree during seed development. PLoS One 7:e43084PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994PubMedCrossRefGoogle Scholar
  10. 10.
    Kim S, Song J, Choi HT (2004) Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration. FEMS Microbiol Lett 233:201–204PubMedCrossRefGoogle Scholar
  11. 11.
    Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: bestkeeper—Excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515PubMedCrossRefGoogle Scholar
  12. 12.
    Qian J, Xu H, Song J, Xu J, Zhu Y, Chen S (2013) Genome-wide analysis of simple sequence repeats in the model medicinal mushroom Ganoderma lucidum. Gene 512:331–336PubMedCrossRefGoogle Scholar
  13. 13.
    Sanodiya BS, Thakur GS, Baghel RK, Prasad GB, Bisen PS (2009) Ganoderma lucidum: a potent pharmacological macrofungus. Curr Pharm Biotechnol 10:717–742PubMedCrossRefGoogle Scholar
  14. 14.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108PubMedCrossRefGoogle Scholar
  15. 15.
    Tasara T, Stephan R (2007) Evaluation of housekeeping genes in Listeria monocytogenes as potential internal control references for normalizing mRNA expression levels in stress adaptation models using real-time PCR. FEMS Microbiol Lett 269:265–272PubMedCrossRefGoogle Scholar
  16. 16.
    Trapnell C, Hendrickson DG, Sauvageau M, Goff L, Rinn JL, Pachter L (2012) Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31:46–53PubMedCrossRefGoogle Scholar
  17. 17.
    Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Wan H, Yuan W, Ruan M, Ye Q, Wang R, Li Z, Zhou G, Yao Z, Zhao J, Liu S, Yang Y (2011) Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper (Capsicum annuum L.). Biochem Biophys Res Commun 416:24–30PubMedCrossRefGoogle Scholar
  19. 19.
    Xie F, Sun G, Stiller JW, Zhang B (2011) Genome-wide functional analysis of the cotton transcriptome by creating an integrated EST database. PLoS ONE 6:e26980PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Xu JW, Xu YN, Zhong JJ (2012) Enhancement of ganoderic acid accumulation by overexpression of an N-terminally truncated 3-hydroxy-3-methylglutaryl coenzyme a reductase gene in the basidiomycete Ganoderma lucidum. Appl Environ Microbiol 78:7968–7976PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Xu Y, Zhu X, Gong Y, Xu L, Wang Y, Liu L (2012) Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR. Biochem Biophys Res Commun 424:398–403PubMedCrossRefGoogle Scholar
  22. 22.
    Yuan JS, Reed A, Chen F, Stewart CN Jr (2006) Statistical analysis of real-time PCR data. BMC Bioinform 7:85CrossRefGoogle Scholar
  23. 23.
    Żyżyńska-Granica B, Koziak K (2012) Identification of suitable reference genes for real-time PCR analysis of statin-treated human umbilical vein endothelial cells. PLoS One 7:e51547PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Jiang Xu
    • 1
    • 3
  • ZhiChao Xu
    • 1
  • YingJie Zhu
    • 1
  • HongMei Luo
    • 1
  • Jun Qian
    • 1
  • AiJia Ji
    • 1
  • YuanLei Hu
    • 1
    • 2
  • Wei Sun
    • 1
    • 3
  • Bo Wang
    • 1
  • JingYuan Song
    • 1
  • Chao Sun
    • 1
  • ShiLin Chen
    • 1
    • 3
  1. 1.The National Engineering Laboratory for Breeding of Endangered Medicinal Materials, Institute of Medicinal Plant DevelopmentChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
  2. 2.State Key Laboratory of Protein and Plant Gene Research, College of Life SciencesPeking University BeijingBeijingChina
  3. 3.Institute of Chinese Materia MedicaChinese Academy of Chinese Medical ScienceBeijingChina

Personalised recommendations