Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 116, Issue 1, pp 55–66 | Cite as

Evaluation of suitable reference genes for qRT-PCR gene expression normalization in reproductive, vegetative tissues and during fruit development in oil palm

  • Wan-Chin Yeap
  • Jia Mayne Loo
  • Yick Ching Wong
  • Harikrishna Kulaveerasingam
Original Paper


Gene expression patterns of a target gene in different tissues and at different stages of development can provide clues towards the understanding of its biological function. The accuracy of gene expression via quantitative real-time reverse transcription-PCR (qRT-PCR) analysis strongly depends on transcript normalization using reference genes that demonstrate consistent expression levels in tissues and experimental conditions studied. However, suitable reference genes for qRT-PCR in oil producing fruit have not been well identified. In this study, the expression stability of fourteen potential reference genes for qRT-PCR analysis in diverse sets of biological samples including six distinct oil palm organs, two floral tissues, four vegetative tissues, five stages of fruit development and two differential phenotype fruit tissue was evaluated. The expression stabilities were assessed using three statistical algorithms including geNorm, NormFinder and qBASE plus. The stability rankings from three outputs were consolidated to obtain the consensus stability ranking of all reference genes. Our analysis showed that a Gibberellin-responsive protein (GRAS) and Cyclophilin 2 (Cyp2) were the most stable genes in oil palm tissues tested. A combination of Cyp2, GRAS and SLU7 were identified as most suitable reference genes for all oil palm tissues and vegetative tissues set while in reproductive tissues set, GRAS and Glutaredoxin was recommended. Cyp2 and GRAS were required for normalization in mesocarp tissues at various developmental stages and differential oil yielding mesocarp tissues. In addition, the relative gene expression profile of two fatty acid biosynthesis genes, EgACCase and EgSTEA was conducted to confirm the validity of the reference genes in this study. Both EgACCase and EgSTEA were analyzed in fruit tissues at various developmental stages. This study identified the most suitable reference genes for normalization of gene expression in oil palm. These results serve as a guideline for the selection of suitable reference genes under different experimental conditions and accurate normalization of gene expression studies in a wide variety of tissues in oil palm.


Quantitative real-time RT-PCR Reference gene Oil palm Normalization Gene expression Fruit development 



This study was financially supported by Sime Darby Plantation. The authors thank SDR Banting for the oil palm samples, Dr. Hamidah Musa and Lee Fong Chin for their helpful suggestions. We also thank the reviewers for their critical comments on the manuscript.

Supplementary material

11240_2013_382_MOESM1_ESM.pdf (75 kb)
Supplementary material 1 (PDF 75 kb)
11240_2013_382_MOESM2_ESM.pdf (76 kb)
Supplementary material 2 (PDF 76 kb)
11240_2013_382_MOESM3_ESM.pdf (94 kb)
Supplementary material 3 (PDF 93 kb)


  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. Asemota O, Shah FH (2004) Detection of mesocarp oleoyl-thioesterase gene of the South American oil palm Elaeis oleifera by reverse transcriptase polymerase chain reaction. Afr J Biotechnol 3:595–598Google Scholar
  3. Barsalobres-Cavallari C, Severino FE, Maluf MP, Maia IG (2009) Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions. BMC Mol Biol 10:1PubMedCentralPubMedCrossRefGoogle Scholar
  4. Bourgis F, Kilaru A, Cao X, Ngando-Ebongue GF, Drira N, Ohlrogge JB, Arondel V (2011) Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning. Proc Natl Acad Sci USA 108:12527–12532PubMedCrossRefGoogle Scholar
  5. Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR RT-PCR: trends and problems. J Mol Endocrinol 29:23–29PubMedCrossRefGoogle Scholar
  6. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622PubMedCrossRefGoogle Scholar
  7. Chandna R, Augustine R, Bisht NC (2012) Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR. PLoS ONE 7:e36918PubMedCentralPubMedCrossRefGoogle Scholar
  8. Chao WS, Dogramaci M, Foley ME, Horvart DP, Anderson JV (2012) Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula). PLoS ONE 7:e42839PubMedCentralPubMedCrossRefGoogle Scholar
  9. Chen D, Pan X, Xiao P, Farwell MA, Zhang B (2011) Evaluation and identification of reliable reference genes for pharmacogenomics, toxicogenomics, and small RNA expression analysis. J Cell Physiol 226:2469–2477PubMedCrossRefGoogle Scholar
  10. Czeschowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17CrossRefGoogle Scholar
  11. Czikkel BE, Maxwell DP (2007) NtGRAS1, a novel stress-induced member of the GRAS family in tobacco, localizes to the nucleus. J Plant Physiol 164:1220–1230PubMedCrossRefGoogle Scholar
  12. Exposito-Rodriguez M, Borges AA, Borges-Perez A, Perez JA (2008) Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol 8:131PubMedCentralPubMedCrossRefGoogle Scholar
  13. Fu J, Wang Y, Huang H, Zhang C, Dai S (2012) Reference gene selection for RT-qPCR analysis of Chrysanthemum lavandulifolium during its flowering stages. Mol Breeding 31:205–215CrossRefGoogle Scholar
  14. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8:R19PubMedCentralPubMedCrossRefGoogle Scholar
  15. Hirsch S, Oldroyd GED (2009) Mini-Review GRAS-domain transcription factors that regulate plant development. Plant Signal Behav 4:698–700PubMedCentralPubMedCrossRefGoogle Scholar
  16. 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 176:702–706CrossRefGoogle Scholar
  17. Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345:646–651PubMedCrossRefGoogle Scholar
  18. Jian B, Liu B, Bi Y, Hou W, Wu C, Han T (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:59PubMedCentralPubMedCrossRefGoogle Scholar
  19. Jose VD, Roman B, Nadal S, Gonzalez-Verdejo CI (2010) Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232:145–153CrossRefGoogle Scholar
  20. Ky H, Thuc LV, Ooi SE, Ishak Z, Namasivayam P, Napis S (2009) Sequence and expression analysis of EgSAPK, a putative member of the serine/threonine protein kinases in oil palm (Elaies guineensis Jacq.). Int J Bot 5:76–84CrossRefGoogle Scholar
  21. Liu Z, Ge XX, Wu XM, Kou SJ, Chai LJ, Guo WW (2013) Selection and validation of suitable reference genes for mRNA qRT-PCR analysis using somatic embryogenic cultures, floral and vegetative tissues in citrus. Plant Cell Tissue Organ Cult 113:s469–s481CrossRefGoogle Scholar
  22. Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines M (2010) Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrid. BMC Plant Biol 10:4PubMedCentralPubMedCrossRefGoogle Scholar
  23. Nakkaew A, Chotigeat W, Eksomtramage T, Phongdara A (2008) Cloning and expression of a plastid-encoded subunit, beta-carboxyltransferase gene (accD) and a nuclear-encoded subunit, biotin carboxylase of acetyl-CoA carboxylase from oil palm (Elaeis guineensis Jacq.). Plant Sci 175:497–504CrossRefGoogle Scholar
  24. Narsai R, Inanova A, Ng S, Whelan J (2010) Defining reference genes in Oryza sativa using organ, development, biotic and abiotic transcriptome datasets. BMC Plant Biol 10:56PubMedCentralPubMedCrossRefGoogle Scholar
  25. Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalisation in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914PubMedCrossRefGoogle Scholar
  26. Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nat Protoc 1:1559–1582PubMedCrossRefGoogle Scholar
  27. Paolacci AR, Tanzarella OA, Porceddu E, Ciaffi M (2009) Identification and validation of reference genes of quantitative RT-PCR normalization in wheat. BMC Mol Biol 10:11PubMedCentralPubMedCrossRefGoogle Scholar
  28. Qi J, Yu S, Zhnag F, Shen X, Zhao X, Yu Y, Zhang D (2010) Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcripts levels in Chinese cabbage (Brassica rapa L.ssp. pikenensis). Plant Mol Biol Rep 28:597–604CrossRefGoogle Scholar
  29. Reid KE, Olsson N, Schlosser J, Peng F, Lund ST (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Methodol 6:27CrossRefGoogle Scholar
  30. Rival A, Jaligot E, Beule T, Jean Finnegan E (2008) Isolation and expression analysis of genes encoding MET, CMT, and DRM methyltransferases in oil palm (Elaeis guineensis Jacq.) in relation to the ‘mantled’ somaclonal variation. J Exp Bot 59:3271–3281PubMedCrossRefGoogle Scholar
  31. Romana PGN, Horton P, Gray JE (2004) The arabidopsis cyclophilin gene family. Plant Physiol 134:1268–1282CrossRefGoogle Scholar
  32. Schmittgen TD, Zakrajsek BA (2002) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46:69–81CrossRefGoogle Scholar
  33. Shearman JR, Jantasuriyarat C, Sangsrakru D, Yoocha T, Vannavichit A, Tragoonrung S, Tangphatsornruang S (2013) Transcriptome analysis of normal and mantled developing oil palm flower and fruit. Genomics 101:306–312PubMedCrossRefGoogle Scholar
  34. Tee SS, Tan YC, Abdullah F, Ong-Abdullah M, Ho CL (2013) Transcriptome of oil palm (Elaeis guineensis Jacq.) roots treated with Ganoderma boninense. Tree Genet Genomes 9:377–386CrossRefGoogle Scholar
  35. Tranbarger TJ, Dussert S, Joët T, Argout X, Summo M, Champion A, Cros D, Omore A, Nouy B, Morcillo F (2011) Regulatory mechanisms underlying oil palm fruit mesocarp maturation, ripening, and functional specialization in lipid and carotenoid metabolism. Plant Physiol 156:564–584PubMedCentralPubMedCrossRefGoogle Scholar
  36. 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:research0034.1–research0034.11Google Scholar
  37. Wang SB, Liu KW, Diao WP, Li Z, Ge W, Liu JB, Pan BG, Wan HJ, Chen JF (2012) Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative real-time PCR. Mol Breeding 30:1393–1400CrossRefGoogle Scholar
  38. Yeap WC, Ooi TEK, Parameswari N, Harikrishna K, Ho CL (2012) EgRBP42 encoding an hnRNP-like RNA binding protein from Elaeis guineensis Jacq. is responsive to abiotic stresses. Plant Cell Rep 31:1829–1843PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Wan-Chin Yeap
    • 1
  • Jia Mayne Loo
    • 1
  • Yick Ching Wong
    • 1
  • Harikrishna Kulaveerasingam
    • 1
  1. 1.Sime Darby Technology Centre Sdn. Bhd., 1st Floor, Block B, UPM-MTDC Technology Centre III, Lebuh SilikonUniversiti Putra MalaysiaSerdangMalaysia

Personalised recommendations