Abstract
Analysis of gene expression using real-time reverse transcription polymerase chain reaction (RT-PCR) requires reference genes to normalize expression values between samples. We have developed a novel reference for real-time RT-PCR using an arbitrary primer to amplify a random set of genes. The arbitrary primer amplifies over 30 genes, whose cumulative expression as measured by real-time RT-PCR closely follows that of UBQ 11, an Arabidopsis thaliana gene that is used as a reference on microarrays. The expression of arbitrary genes is also compared with potato (Solanum tuberosum spp. tubersosum) housekee** genes and was shown to be stable during Phytophthora infestans infection.
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Abbreviations
- DNA:
-
deoxyribonucleic acid
- MoMLV:
-
Moloney murine leukemia virus
- RNA:
-
ribonucleic acid
- RT-PCR:
-
reverse transcription polymerase chain reaction
- DDRT-PCR:
-
differential display reverse-transcription polymerase chain reaction
References
Bower NI, Moser RJ, Hill JR, Lehnert SA (2007) Universal reference method for real-time PCR gene expression analysis of preimplantation embryos. Biotechniques 42:199–206 doi:10.2144/000112314
Bustin SA, Benes V, Nolan T, Pfaffl MW (2005) Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 34:597–601 doi:10.1677/jme.1.01755
Caetano-Anolles G (1993) Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl 3:85–94
Czechowski T, Bari RP, Stitt M, Scheible WR, Udvardi MK (2004) Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J 38:366–379 doi:10.1111/j.1365-313X.2004.02051.x
Czechowski 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 139:5–17 doi:10.1104/pp.105.063743
Dallas PB, Gottardo NG, Firth MJ, Beesley AH, Hoffmann K, Terry PA et al (2005) Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR—how well do they correlate? BMC Genomics 6:59 doi:10.1186/1471-2164-6-59
Evers D, Schweitzer C, Nicot N, Gigliotti S, Herrera MR, Hausman JF et al (2006) Two PR-1 loci detected in the native cultivated potato Solanum phureja appear differentially expressed upon challenge by late blight. Physiol Mol Plant Pathol 67:155–163 doi:10.1016/j.pmpp.2005.12.003
Galbraith DW, Birnbaum K (2006) Global studies of cell type-specific gene expression in plants. Annu Rev Plant Biol 57:451–475 doi:10.1146/annurev.arplant.57.032905.105302
Gallardo K, Job C, Groot SPC, Puype M, Demol H, Vandekerckhove J et al (2001) Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiol 126:835–848 doi:10.1104/pp.126.2.835
Higuchi R, Dollinger G, Walsh PS, Griffith R (1992) Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY) 10:413–417 doi:10.1038/nbt0492-413
Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971 doi:10.1126/science.1354393
McClelland M, Mathieu-Daude F, Welsh J (1995) RNA fingerprinting and differential display using arbitrarily primed PCR. Trends Genet 11:242–246 doi:10.1016/S0168-9525(00)89058-7
Nakabayashi K, Okamoto M, Koshiba T, Kamiya Y, Nambara E (2005) Genome-wide profiling of stored mRNA in Arabidopsis thaliana seed germination: epigenetic and genetic regulation of transcription in seed. Plant J 41:697–709 doi:10.1111/j.1365-313X.2005.02337.x
Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekee** gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914 doi:10.1093/jxb/eri285
Provenzano M, Mocellin S (2007) Complementary techniques: validation of gene expression data by quantitative real time PCR. Adv Exp Med Biol 593:66–73 doi:10.1007/978-0-387-39978-2_7
Quackenbush J (2002) Microarray data normalization and transformation. Nat Genet 32(Suppl):496–501 doi:10.1038/ng1032
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 doi:10.1016/j.bbrc.2003.11.177
Reid K, Olsson N, Schlosser J, Peng F, Lund S (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6:27 doi:10.1186/1471-2229-6-27
Smith RD, Brown B, Ikonomi P, Schechter AN (2003) Exogenous reference RNA for normalization of real-time quantitative PCR. Biotechniques 34:88–91
Tai HH, Tai GCC, Beardmore T (2005) Dynamic histone acetylation of late embryonic genes during seed germination. Plant Mol Biol 59:909–925 doi:10.1007/s11103-005-2081-x
Tai HH, Williams M, Iyengar A, Yeates J, Beardmore T (2007) Regulation of the β-hydroxyacyl ACP dehydratase gene of Picea mariana by alternative splicing. Plant Cell Rep V26:105
Venkatesh B, Hettwer U, Koopmann B, Karlovsky P (2005) Conversion of cDNA differential display results (DDRT-PCR) into quantitative transcription profiles. BMC Genomics 6:51 doi:10.1186/1471-2164-6-51
Wan CY, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem 223:7–12 doi:10.1006/abio.1994.1538
Wang Y, Barbacioru C, Hyland F, **ao W, Hunkapiller KL, Blake J et al (2006) Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays. BMC Genomics 7:59 doi:10.1186/1471-2164-7-59
Acknowledgments
The authors would like to thank Ian Macdonald who provided technical assistance for this study.
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Tai, H.H., Conn, G., Davidson, C. et al. Arbitrary Multi-gene Reference for Normalization of Real-Time PCR Gene Expression Data. Plant Mol Biol Rep 27, 315–320 (2009). https://doi.org/10.1007/s11105-009-0089-0
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DOI: https://doi.org/10.1007/s11105-009-0089-0