Hostname: page-component-cd9895bd7-7cvxr Total loading time: 0 Render date: 2024-12-23T12:20:42.755Z Has data issue: false hasContentIssue false

Identification and validation of reference genes for gene expression studies in water buffalo

Published online by Cambridge University Press:  05 February 2010

V. Terzi*
Affiliation:
C.R.A., Genomic Research Centre, Via San Protaso 302, 29017-Fiorenzuola d’Arda, Italy
C. Morcia
Affiliation:
C.R.A., Genomic Research Centre, Via San Protaso 302, 29017-Fiorenzuola d’Arda, Italy
M. Spini
Affiliation:
C.R.A., Genomic Research Centre, Via San Protaso 302, 29017-Fiorenzuola d’Arda, Italy
R. Tudisco
Affiliation:
Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, sez. B. Ferrara, Università di Napoli Federico II, Via Delpino 1, 80055-Napoli, Italy
M. I. Cutrignelli
Affiliation:
Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, sez. B. Ferrara, Università di Napoli Federico II, Via Delpino 1, 80055-Napoli, Italy
F. Infascelli
Affiliation:
Dipartimento di Scienze Zootecniche e Ispezione degli Alimenti, sez. B. Ferrara, Università di Napoli Federico II, Via Delpino 1, 80055-Napoli, Italy
A. M. Stanca
Affiliation:
C.R.A., Genomic Research Centre, Via San Protaso 302, 29017-Fiorenzuola d’Arda, Italy
P. Faccioli
Affiliation:
C.R.A., Genomic Research Centre, Via San Protaso 302, 29017-Fiorenzuola d’Arda, Italy
*
Get access

Abstract

In gene expression analysis, a key step to obtain informative data from reverse transcription quantitative PCR (RT qPCR) assay is normalization, that is usually achieved by ratio to correct the abundance of the gene of interest against that of an endogenous reference gene. The finding of such reference genes, ideally expressed in a stable way in multiple tissue samples and in different experimental conditions, is a non-trivial problem. In this work, a set of genes potentially useful as reference for gene expression studies in water buffalo has been identified and evaluated. In the first step, a publicly available Bos taurus expressed sequence tags database has been downloaded from the TIGR Gene Index and mined by some simple frequency algorithms to find out which tentative consensuses are present in a remarkable number of different cDNA libraries and, consequently, are more suitable to be included in a starter set of candidate reference genes. To validate the potential of such candidates for their use as normalizers in buffalo gene expression analysis, an RT qPCR analysis has been carried out, in which the expression stability of these genes has been evaluated on a panel of buffalo tissues and organs. Our results indicate that ribosomal proteins L4 and L5 and Gek protein encoding genes can be useful as normalizers to compare gene expression levels across tissues and organs in buffalo.

Type
Full Paper
Copyright
Copyright © The Animal Consortium 2010

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abruzzo, LV, Lee, KY, Fuller, A, Silverman, A, Keating, MJ, Medeiros, LJ, Coombes, KR 2005. Validation of oligonucleotide microarray data using microfluidic low-density arrays: a new statistical method to normalize real-time RT-PCR data. BioTechniques 38, 785792.CrossRefGoogle ScholarPubMed
Andersen, CL, Jensen, JL, Ørntaft, TF 2004. Normalization of real-time quantitative reverse transcryptic-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research 64, 52455250.CrossRefGoogle Scholar
Aswal, APS, Datta, TK, Raghav, S, De, S, Yadav, P, Goswani, SL 2007. Development of a competitive quantitative PCR strategy for evaluating the expression stability of 18s rRNA during in vitro maturation of buffalo (Bubalus bubalis) follicular oocytes. Reproduction in Domestic Animals 42, 195201.CrossRefGoogle ScholarPubMed
Aswal, APS, Raghav, S, De, S, Yadav, P, Thakur, M, Goswani, SL, Datta, TK 2008. Expression stability of two reference genes (18s rRNA and G3PDH) during in vitro maturation of follicular oocytes in buffalo (Bubalus bubalis). Animal Reproduction Science 103, 164171.CrossRefGoogle Scholar
Ayers, D, Clemens, DN, Salway, F, Day, PJR 2007. Expression stability of commonly used reference genes in canine articular connective tissues. BMC Veterinary Research 3, 7.CrossRefGoogle ScholarPubMed
Bettegowda, A, Patel, OV, Ireland, JJ, Smith, GW 2006. Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro. Molecular Reproductive Development 73, 267278.CrossRefGoogle ScholarPubMed
Bionaz, M, Loor, JJ 2007. Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during lactation cycle. Physiological Genomics 29, 312319.CrossRefGoogle ScholarPubMed
Bustin, SA 2002. Quantitation of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Journal of Molecular Endocrinology 29, 2339.CrossRefGoogle Scholar
Czechowski, T, Stitt, M, Altmann, T, Udvardi, MK, Scheible, W 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiology 139, 517.CrossRefGoogle ScholarPubMed
De Ketelaere, A, Goossens, K, Peelman, L, Burvenich, C 2006. Technical note: validation of internal control genes for gene expression analysis in bovine polymorphonuclear leukocytes. Journal of Dairy Science 89, 40664069.CrossRefGoogle ScholarPubMed
Ding, C, Cantor, CR 2004. Quantitative analysis of nucleic acids-the last few years of progress. Journal of Biochemical Molecular Biology 37, 110.Google ScholarPubMed
Donaldson, L, Vuocolo, T, Gray, C, Strandberg, Y, Reverter, A, McWilliam, S, Wang, Y-H, Byrne, K, Tellam, R 2005. Construction and validation of a bovine innate immune microarray. BMC Genomics 6, 135.CrossRefGoogle ScholarPubMed
Faccioli, P, Ciceri, GP, Provero, P, Stanca, AM, Morcia, C, Terzi, V 2007. 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 Molecular Biology 63, 679688.CrossRefGoogle ScholarPubMed
Feng, S, Salter, AM, Parr, T, Gransworthy, PC 2007. Extraction and quantitative analysis of stearoyl-coenzyme A desaturase mRNA from dairy cow milk somatic cells. Journal of Dairy Science 90, 41284136.CrossRefGoogle ScholarPubMed
Garcia-Crespo, D, Juste, RA, Hurtado, A 2005. Selection of ovine reference genes for normalisation by real-time RT-PCR; analysis of PrP gene expression and genetic susceptibility to scrapie. BMC Veterinary Research 1, 3.CrossRefGoogle Scholar
Goossens, K, Van Poucke, M, Van Soom, A, Vandesompele, J, Van Zeveren, A, Peelman, LJ 2005. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Developmental Biology 5, 27.CrossRefGoogle ScholarPubMed
Hocquette, J-F, Brandstetter, AM 2002. Common practice in molecular biology may introduce statistical bias and misleading biological interpretation. Journal of Nutritional Biochemistry 13, 370377.CrossRefGoogle ScholarPubMed
Huggett, J, Dheda, K, Bustin, S, Zumla, A 2005. Real-time RT-PCR normalisation; strategies and considerations. Gene and Immunity 6, 279284.CrossRefGoogle ScholarPubMed
Khatib, H, Schutzkus, V 2006. The expression profile of the H19 gene in cattle. Mammalian Genome 17, 991996.CrossRefGoogle ScholarPubMed
Kubista, M, Andrade, JM, Bengtsson, M, Forootan, A, Jonák, J, Lind, K, Sindelka, R, Sjőback, , Sjőgreen, B, Strőmbom, L, Ståhlberg, A, Zoric, N 2006. The real-time polymerase chain reaction. Molecular Aspects of Medicine 27, 95125.CrossRefGoogle ScholarPubMed
Janovick-Guretzy, NA, Dann, HM, Carlson, DB, Murphy, MR, Loor, JJ, Drackley, JK 2007. Reference gene expression in bovine liver is affected by physiological state, feed intake and dietary treatment. Journal of Dairy Science 90, 22462252.CrossRefGoogle Scholar
Jones, LJ, Yue, ST, Cheung, C, Singer, VL 1998. RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization. Analytical Biochemistry 265, 368374.CrossRefGoogle ScholarPubMed
Lee, PD, Sladek, R, Greenwood, CMT, Hudson, TJ 2002. Control genes and variability: absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Research 12, 292297.CrossRefGoogle ScholarPubMed
Lisowski, P, Pierzchala, M, Goscik, J, Pareek, CS, Zweirzchowski, L 2008. Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid. Journal of Applied Genetics 49, 367372.CrossRefGoogle Scholar
Navani, N, Jain, PK, Gupta, S, Sisodia, BS 2002. A set of cattle microsatellite DNA markers for genome analysis of riverine buffalo (Bubalus bubalis). Animal Genetics 33, 1493154.CrossRefGoogle ScholarPubMed
Nicot, N, Hausman, J, Hoffmann, L, Evers, D 2005. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany 56, 29072914.CrossRefGoogle ScholarPubMed
Nolan, T, Hands, RE, Bustin, SA 2006. Current problems in quantitative real-time RT-PCR. Nature Protocols 1, 15591582.CrossRefGoogle Scholar
Pfaffl, MW 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acid Research 29, e45.CrossRefGoogle ScholarPubMed
Pfaffl, MW, Lange, IG, Daxenberger, A, Meyer, HHD 2001. Tissue-specific expression pattern of estrogen receptors (ER): quantification of ER alpha and ER beta mRNA with real-time RT-PCR. APMIS 109, 345355.CrossRefGoogle ScholarPubMed
Radonić, A, Thulke, S, Mackay, IM, Landt, O, Siegert, W, Nitsche, A 2004. Guidelines to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications 313, 856862.CrossRefGoogle Scholar
Ritz, LR, Glowatzki-Mullis, ML, MacHugh, DE, Gaillard, C 2000. Phylogenetic analysis of the tribe Bovini using microsatellites. Animal Genetics 31, 178185.CrossRefGoogle ScholarPubMed
Robinson, TL, Sutherland, IA, Sutherland, J 2007. Validation of candidate bovine reference genes for use with real-time PCR. Veterinary Immunology and Immunopathology 115, 160165.CrossRefGoogle ScholarPubMed
Schmittgen, TD, Zakrajsek, BA 2000. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. Journal of Biochemical and Biophysical Methods 46, 6981.CrossRefGoogle ScholarPubMed
Schroeder, A, Mueller, O, Stocker, S, Salowsky, R, Leiber, M, Gassmann, M, Lightfoot, S, Menzel, W, Granzow, M, Ragg, T 2006. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology 7, 3.CrossRefGoogle ScholarPubMed
Stafuzza, NB, Ianella, P, Miziana, MN, Agarwala, R, Schäffer, AA, Riggs, PK, Womack, JE, Amaral, MEJ 2007. Preliminary radiation hybrid map for river buffalo chromosome 6 and comparison to bovine chromosome 3. Animal Genetics 38, 406409.CrossRefGoogle ScholarPubMed
Vandesompele, J, De Praeter, 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 Biology 3 research 0034.11.CrossRefGoogle ScholarPubMed
Warrington, J, Nair, A, Mahadevappa, M, Tsyganskaya, M 2000. Comparison of human adult and fetal expression and identification of 535 housekeeping/maintenance genes. Physiological Genomics 2, 143147.CrossRefGoogle ScholarPubMed