Identification and validation of reference genes for quantitative RT-PCR normalization in wheat
Tóm tắt
Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple
The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin.
The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.
Từ khóa
Tài liệu tham khảo
Czechowski T, Bari RP, Stitt M, Scheible W-R, Udvardi MK: Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J. 2004, 38: 366-379. 10.1111/j.1365-313X.2004.02051.x
Gachon C, Mingam A, Charrier B: Real-time PCR: what relevance to plant studies?. J Exp Bot. 2004, 55: 1445-1454. 10.1093/jxb/erh181
Caldana C, Scheible W-R, Mueller-Roeber , Ruzicic S: A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors. Plant Methods. 2007, 3: 7- 10.1186/1746-4811-3-7
Bustin SA: Quantification of mRNA using real-time reverse transcription PCR RT-PCR: trends and problems. J Mol Endocrinol. 2002, 29: 23-29. 10.1677/jme.0.0290023
Ginzinger DG: Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp Hematol. 2002, 30 (6): 503-512. 10.1016/S0301-472X(02)00806-8
Huggett J, Dheda K, Bustin S, Zumla A: Real-time RT-PCR normalisation; strategies and considerations. Genes Immun. 2005, 6 (4): 279-284. 10.1038/sj.gene.6364190
Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Henne G, Grisar T, Igout A, Heinen E: Housekeeping genes as internal standards: use and limits. J Biotechnol. 1999, 75 (2-3): 191-195. 10.1016/S0168-1656(99)00163-7
Suzuki T, Higgins PJ, Crawford DR: Control selection for RNA quantitation. Biotechniques. 2000, 29: 332-337.
Selvey S, Thompson EW, Matthaei K, Lea RA, Irving MG, Griffiths LR: Beta-actin an unsuitable internal control for RT-PCR. Mol Cell Probes. 2001, 15 (5): 307-311. 10.1006/mcpr.2001.0376
Lee PD, Sladek R, Greenwood CMT, Hudson TJ: Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res. 2001, 12: 292-297. 10.1101/gr.217802.
Ohl F, Jung M, Xu C, Stephan C, Rabien A, Burkhardt M, Nitsche A, Kristiansen G, Loening SA, Radonic A, Jung K: Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?. J Mol Med. 2005, 83 (12): 1014-1024. 10.1007/s00109-005-0703-z
Czechowski T, Stitt M, Altman T, Udvardi MK, Scheible W-R: Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 2005, 139 (1): 5-17. 10.1104/pp.105.063743
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, 3 (7): RESEARCH0034- 10.1186/gb-2002-3-7-research0034
Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004, 26 (6): 509-515. 10.1023/B:BILE.0000019559.84305.47
Andersen CL, Jensen JL, Orntoft 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. Cancer Res. 2004, 64 (15): 5245-5250. 10.1158/0008-5472.CAN-04-0496
Brunner AM, Yakovlev IA, Strauss SH: Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol. 2004, 4: 14- 10.1186/1471-2229-4-14
Goncalves S, Cairney J, Maroco J, Oliveira MM, Miguel C: Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis. Planta. 2005, 222: 556-563. 10.1007/s00425-005-1562-0
Nicot N, Hausman JF, Hoffmann L, Evers D: Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot. 2005, 56: 2907-2914. 10.1093/jxb/eri285
Jain M, Nijhawan A, Tyagi AK, Khurana JP: Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Com. 2006, 345: 646-651. 10.1016/j.bbrc.2006.04.140
Reid KE, Olsson N, Schlosser J, Peng F, Lund ST: An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol. 2006, 6: 27- 10.1186/1471-2229-6-27
Faccioli P, Ciceri GP, Provero P, Stanca AM, Morcia C, Terzi V: A combined strategy of "in silico" transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies. Plant Mol Biol. 2007, 63: 679-688. 10.1007/s11103-006-9116-9
Gutierrez L, Mauriat M, Pelloux J, Bellini C, Van Wuytswinkel O: Towards a systematic validation of references in Real-Time RT-PCR. Plant Cell. 2008, 20: 1734-1735. 10.1105/tpc.108.059774
Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefevre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O: The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnology J. 2008, 6: 609-618. 10.1111/j.1467-7652.2008.00346.x.
Feuillet C, Keller B: Comparative genomics in the grass family: molecular characterization of grass genome structure and evolution. Ann Bot. 2002, 89: 3-10. 10.1093/aob/mcf008
La Rota M, Sorrells ME: Comparative DNA sequence analysis of mapped wheat ESTs reveals the complexity of genome relationships between rice and wheat. Funct Integr Genomics. 2004, 4: 34-46. 10.1007/s10142-003-0098-2
Singh NK, Dalal V, Batra K, Singh BK, Chitra G, et al: Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes. Funct Integr Genomics. 2007, 7: 17-35. 10.1007/s10142-006-0033-4
Crismani W, Baumann U, Sutton T, Shirley N, Webster T, Spangenberg G, Langridge P, Able JA: Microarray expression analysis of meiosis and microsporogenesis in hexaploid bread wheat. BMC Genomics. 2006, 7: 267- 10.1186/1471-2164-7-267
Wan Y, Poole RL, Huttly AK, Toscano-Underwood C, Feeney K, Welhan S, et al: Transcriptome analysis of grain development in hexaploid wheat. BMC genomics. 2008, 9: 121- 10.1186/1471-2164-9-121
Xue GP, McIntyre CL, Jenkins CLD, Glassop D, van Herwaarden AF, Shorter R: Molecular dissection of variation in carbohydrate metabolism related to water-soluble carboydrate accumulation in stems of wheat. Plant Physiol. 2008, 146: 441-454. 10.1104/pp.107.113076
Clarke B, Rahman S: A microarray analysis of wheat grain hardness. Theor Appl Genet. 2005, 110: 1259-1267. 10.1007/s00122-005-1962-3
Kawaura K, Mochida K, Yamazaki Y, Ogihara Y: Transcriptome analysis of salinity stress responses in common wheat using a 22k oligo-DNA microarray. Funct Integr Genomics. 2006, 6: 132-142. 10.1007/s10142-005-0010-3
Gregersen PL, Holm PB: Transcriptome analysis of senescence in the flag leaf of wheat (Triticum aestivum L.). Plant Biotechnol J. 2007, 5: 192-206. 10.1111/j.1467-7652.2006.00232.x
Bustin SA, Benes V, Nolan T, Pfaffl MW: Quantitative real-time RT-PCR – a perspective. J Mol Endocrinol. 2005, 34: 597-601. 10.1677/jme.1.01755
Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A: Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Com. 2004, 313: 856-862. 10.1016/j.bbrc.2003.11.177
McDowell JM, Huang S, McKinney EC, An Y-Q, Meagher RB: Structure and evolution of the actin gene family in Arabidopsis thaliana. Genetics. 1996, 142: 587-602.
Farajalla MR, Gulick PJ: The α-tubulin gene family in wheat (Triticum aestivum L.) and differential gene expression during cold acclimation. Genome. 50: 502-510.
Wheeler DL, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, Church DM, DiCuccio M, Edgar R, Federhen S, Feolo M, et al: Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2007, 36: 13-21. 10.1093/nar/gkm1000.
Quackenbush J, Cho J, Lee D, Liang F, Holt I, Karamycheva S, Parvizi V, Pertea G, Sultana R, White J: The TIGR Gene Indices: analysis of gene transcript sequences in higly sampled eukaryotic species. Nucleic Acids Res. 29: 159-164.
Ciaffi M, Paolacci AR, Dominici L, Tanzarella OA, Porceddu E: Molecular characterization of gene sequences coding for protein disulfide isomerase (PDI) in durum wheat (Triticum turgidum ssp durum). Gene. 2001, 265: 147-156. 10.1016/S0378-1119(01)00348-1
Ciaffi M, Paolacci AR, D'Aloisio E, Tanzarella OA, Porceddu E: Cloning and characterization of wheat PDI (protein disulfide isomerase) homoeologous genes and promoter sequences. Gene. 2006, 366: 209-218. 10.1016/j.gene.2005.07.032
Tsvetanov S, Ohno R, Tsuda K, Takumi S, Mori N, Atanassov A, Nakamura C: A cold-responsive wheat (Triticum aestivum L.) gene wcor14 identified in a winter-hardy cultivar "Mironovska 808". Genes Genet Syst. 2000, 75: 49-57. 10.1266/ggs.75.49
Ndong C, Danyluk J, Wilson KE, Pocok T, Huner NPA, Sarhan F: Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses. Plant Physiol. 2002, 129: 1368-1381. 10.1104/pp.001925
Campbell JL, Klueva NY, Zheng HG, Nieto-Sotelo J, Ho THD, Nguyen HT: Cloning of new members of heat shock protein HSP101 gene family in wheat (Triticum aestivum L. Moench) inducible by heat, dehydration, and ABA. Biochimica et Biophysica Acta. 2001, 1517: 270-277.
Gulli M, Corradi M, Rampino P, Marmiroli N, Perrotta C: Four members of the HSP101 gene family are differentially regulated in Triticum durum Desf. FEBS Letters. 2007, 581: 4841-4849. 10.1016/j.febslet.2007.09.010
Katiyar-Agarwal S, Agarwal M, Gallie DR, Grover A: Search for cellular functions of plant Hsp100/Clp family proteins. Crit Rev Plant Sci. 2001, 20: 277-295. 10.1016/S0735-2689(01)80043-5.
Wells DR, Tanguay RL, Le H, Gallie DR: HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status. Genes Dev. 1998, 20: 3236-3251. 10.1101/gad.12.20.3236.
geNorm manual. http://medgen.ugent.be/~jvdesomp/genorm/geNorm_manual.pdf
Hsiao L-L, Dangond F, Yosida T, Hong R, Jensen RV, Misra J, Dillon W, Lee KF, Clark KE, et al: A compendium of gene expression in normal human tissues. Physiol Genomics. 2001, 7: 97-104.
Eisenberg E, Levanon EY: Human housekeeping genes are compact. Trends in Genetics. 2003, 7: 362-365. 10.1016/S0168-9525(03)00140-9.
Zhu J, He F, Song S, Wang J, Yu J: How many human genes can be defined as housekeeping with current expression data?. BMC genomics. 2008, 9: 172- 10.1186/1471-2164-9-172
Wong ML, Medrano JF: Real-time PCR for mRNA quantification. Biotechniques. 2005, 39: 75-85. 10.2144/05391RV01
Charrier B, Champion A, Henry Y, Kreis M: Expression profiling of the whole Arabidopsis shaggy-like kinase multigene family by real-time reverse transcriptase-polymerase chain reaction. Plant Physiol. 2002, 130: 577-590. 10.1104/pp.009175
Jian B, Liu B, Bi Y, Hou W, Wu C, Han T: Validation of internal control for gene expression study in soybean by quantitative real time PCR. BMC Molecular Biology. 2008, 9: 59- 10.1186/1471-2199-9-59
Lyng MB, Laenkholm A-V, Pallisgaard N, Ditzel HJ: Identification of genes for normalization of real-time RT-PCR data in breast carcinomas. BMC Cancer. 2008, 8: 20- 10.1186/1471-2407-8-20
Szabo A, Perou CM, Karaca M, Perreard L, Quackenbush JF, Bernard PS: Statistical modelling for selecting housekeeper genes. Genome Biol. 2004, 5: R59- 10.1186/gb-2004-5-8-r59
Haller F, Kulle B, Schwager S, Gunawan B, von HA, Sultmann H, Fuzesi L: Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization. Anal Biochem. 2004, 335: 1-9. 10.1016/j.ab.2004.08.024
Maccoux LJ, Clements DN, Salway F, Day PJR: Identification of new reference genes for the normalization of canine osteoarthritic tissue transcripts from microarray data. BMC Molecular Biology. 2007, 8: 62- 10.1186/1471-2199-8-62
Hibbeler S, Scharsack JP, Becker S: Housekeeping genes for quantitative expression studies in the three-spined stickleback Gasterosteus aculeatus. BMC Molecular Biology. 2008, 9: 18- 10.1186/1471-2199-9-18
Bonefeld BE, Elfving B, Wegener G: Reference genes for normalization: a study of rat brain tissue. SYNAPSE. 2008, 63: 302-309. 10.1002/syn.20496.
Yu H, Luscombe NM, Qian J, Gerstein M: Genomic analysis of gene expression relationships in transcriptional regulatory networks. Trends Genet. 2003, 19: 422-427. 10.1016/S0168-9525(03)00175-6
Ransbotin V, Reusch TBH: Housekeeping gene selection for quantitative real-time PCR assays in the seagrass Zostera marina subjected to heat stress. Limnology and Oceanography-Methods. 2006, 4: 367-373.
Schmid H, Cohen CD, Henger A, Irrgang S, Schlondoff D, Kretzler M: Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies. Kidney Int. 2003, 64: 356-360. 10.1046/j.1523-1755.2003.00074.x