Abstract
Quantitative real-time polymerase chain reaction (qRT-PCR) is currently the most sensitive method used for quantitative gene expression studies. However, minimal variation in the amount of material and presence of inhibitors affecting enzyme efficiency can lead to significant quantification errors. Accurate data normalization is vital using reference genes as internal controls. Many so-called housekeeping genes or reference genes with assumed stable expression can exhibit either up- or downregulation depending on the developmental stage or other environmental conditions. We have evaluated six reference genes (actin, APRT, 18S rRNA, ef1α, β-tubulin and ribosomal protein L2) for qRT-PCR profiling experiments in potato tuber tissues of five varieties during cold storage at different temperatures and treatment periods. Genes were ranked according to their expression stability by BestKeeper, geNorm and NormFinder software tools in the same order. This means that any of them can be used for this purpose. The results indicated that ef1α and APRT were the most stably expressed genes in the potato tuber tissues under different cold storage regimes. We therefore recommend use of this pair of genes as internal controls for gene expression studies under the described conditions.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andersen CL, Jensen JL, Orntoft TF (2004) Normalization of quantitative real-time PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193
Bustin SA, Mueller R (2005) Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin Sci 109:365–379
Bustin SA, Gyselman VG, Siddiqi S, Dorudi S (2000) Cytokeratin 20 is not a tissue specific marker for the detection of malignant epithelial cells in the blood of colorectal cancer patients. Int J Surg Invest 2:49–57
Caldana C, Scheible WR, Mueller-Roeber B, Ruzicic S (2007) A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors. Plant Methods 3:7. doi:10.1186/1746-4811-3-
Dean JD, Goodwin PH, Hsiang T (2002) Comparison of relative RT-PCR and northern blot analyses to measure expression of β-1,3-glucanasa in Nicatiana benthamiana infected with Colletotrichum destructivum. Plant Mol Biol Rep 20:347–356
Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GA, Zumla A (2005) The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal Biochem 344(1):141–143
Gachon C, Mingam A, Charrier B (2004) Real-Time PCR: what relevance to plant studies. J Exp Bot 55:1445–1454
Gutierrez L, Mauriat M, Pelloux J, Bellini C, Van Wuytswinkel O (2008) Towards a systematic validation of references in real-time RT-PCR. Plant Cell 20:1734–1735
Haase NU (2007) The canon of potato science: 48. Maillard reaction. Potato Res 50:407–409
Hellemans J, Mortier G, De Paepe A, Speleman F, Vandersompele J (2007) qBase relative quantification framework and software for management and automated analysis of real.time quantitative PCR data. Genome Biol 8:R19
Hogervorst JG, Schouten LJ, Konings EJ, Goldbohm RA, van den Brandt PA (2007) A prospective study of dietary acrylamide intake and the risk of endometrial, ovarian, and breast cancer. Cancer Epidemiol Biomarkers Prev 16:2304–2313
Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, Untergasser A, Vandersompele J (2009) RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res 37(7):2065–2069
Mehta R, Birerdinc A, Hossain N, Afendy A, Chandhoke V (2010) Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples. BMC Mol Biol 11(39):1471–2199
Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914
Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time PCR. Nat Protoc 1:1559–1582
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002–2007
Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:e36
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 26:509–515
Pinhero RG, Coffin R, Yada RY (2009) Post-harvest storage in potatoes. In: Jaspreet S, Lovedeep K (eds) Potato Chemistry and Technology. Academic, San Diego
Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:856–862
Ritter E, Ruiz de Galarreta JI, van Eck HJ, Sánchez I (2008) Construction of a transcriptome map of potato based on the cDNA-AFLP technique. Theor Appl Genet 116:1003–1013
Van Es A, Hartmans KJ (1987) Starch and sugars during tuberization, storage and sprouting. In: Rastovski A, Van Es A (eds) Storage of potatoes. PUDOC, Wageningen, pp 141–147
Vandersompele 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
Vandersompele J, Kubista M, Pfaffl W (2009) Reference gene validation software for improved normalization. In: Logan J, Edwards K, Saunders N (eds) Real Time PCR: Current Technology and Applications. Caister Academic, Norfolk
Walker NJ (2002) A technique whose time has come. Science 296:557–559
Willems E, Mateizel I, Kemp C, Cauffman G, Sermon K, Leyns L (2006) Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells. Int J Dev Biol 50(7):627–635
Acknowledgments
The authors especially thank Dr. Cristina Lacasta for technical assistance. Part of this work was financed within the project INIA RTA2011-00018-C03-02.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lopez-Pardo, R., Ruiz de Galarreta, J.I. & Ritter, E. Selection of housekeeping genes for qRT-PCR analysis in potato tubers under cold stress. Mol Breeding 31, 39–45 (2013). https://doi.org/10.1007/s11032-012-9766-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11032-012-9766-z