Estabilidade de genes de referência no músculo Longissimus thoracis et lumborum de ovelhas crioulas colombianas

Autores

DOI:

https://doi.org/10.5433/1679-0359.2022v43n3p987

Palavras-chave:

cDNA, Correlação de pares, Normalização, Quantificação relativa, RT-qPCR.

Resumo

A raça de ovinos Crioula Colombiana tem grande importância econômica e social para a Colômbia. Avaliar a produção e a qualidade da carne de machos e fêmeas é importante para pequenos produtores do país e, assim, faz necessário o uso ferramentas que ajudam a avaliar os pontos críticos de produção, como a reação em cadeia da polimerase em tempo real (Real-time quantitative polymerase chain reaction [RT-qPCR]). Esta é uma ferramenta molecular amplamente usada para a quantificação relativa de genes candidatos em vários tecidos. Para o seu uso correto, é necessário o uso de genes com expressão estável denominados genes de referência. No entanto, estudos recentes têm mostrado que a expressão desses genes de referência pode variar entre os tecidos e pode ser modulada por raça, sexo ou estímulos externos. Da mesma forma, existem poucas informações sobre a expressão desses genes no músculo Longissimus thoracis et lumborum de ovinos machos e fêmeas da raça Crioula Colombiana. Neste estudo foi comparada a estabilidade na expressão de sete genes de referência (ACTB, YWHAZ, SDHA, GAPDH, TUBB2A, B2M e PGK1) no músculo Longissimus thoracis et lumborum de ovinos Crioulo Colombiano machos e fêmeas, por serem genes utilizados em estudos de RT-qPCR visando determinar os mais estáveis para esta raça.Doze animais com peso corporal de 26 ± 4 kg e 12 ± 3 meses de idade foram utilizados em condições de pastejo. Foram realizadas biópsias do músculo Longissimus thoracis et lumborum, de onde o RNA foi extraído e o cDNA foi sintetizado. A expressão foi determinada usando RT-qPCR e sua estabilidade foi analisada por algoritmos computacionais usando geNorm, Normfinder e BestKeeper pacote de software, os quais foram integrados usando RefFinder pacote de software. Os resultados indicam que GAPDH, ACTB e SDHA apresentam maior estabilidade, enquanto a expressão mais variável foi para B2M. Esses dados fornecem a base para resultados mais precisos em estudos de RT-qPCR de expressão gênica em músculos defeminino ovinos da raça Crioula Colombiana.

Métricas

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Biografia do Autor

Edwin Sandoval Lozano, University of Tolima

PhD, Research Group in Livestock Agroforestry Systems, Faculty of Veterinary Medicine and Zootechnics, Department of Livestock Production, University of Tolima, Ibagué, Tolima, Colombia.

Iang Schroniltgen Rondon Barragan, University of Tolima

PhD, Research Group in Immunobiology and Pathogenesis, Faculty of Veterinary Medicine and Zootechnics, Department of Animal Health, University of Tolima, Ibagué, Tolima, Colombia.

Referências

An, Y., Reimers, K., Allmeling, C., Liu, J., Lazaridis, A., & Vogt, P. M. (2012). Validation of differential gene expression in muscle engineered from rat groin adipose tissue by quantitative real-time PCR. Biochemical and Biophysical Research Communications, 421(4), 736-742. doi: 10.1016/j.bbrc.2012.04.073

Ángel, S., & Ramírez, A. (2014). Estudio zoométrico del Ovino de Pelo Criollo Colombiano. AICA, 4(1), 338-340.

Argyropoulos, C. P., Chen, S. S., Ng, Y., Roumelioti, M., Michael, M., & Edey, J. (2017). Rediscovering Beta-2 Microglobulin as a biomarker across the Spectrum of kidney diseases. Frontiers in Medicine, 4(73), 1-25. doi: 10.3389/fmed.2017.00073

Aurélio, M., Fortes, S., Marzuca-nassr, G. N., Vitzel, K. F., Hermano, C., Newsholme, P., & Curi, R. (2016). Housekeeping proteins: how useful are they in skeletal muscle diabetes studies and muscle hypertrophy models? Analytical Biochemistry, 504(1), 38-40. doi: 10.1016/j.ab.2016.03.023

Aziziyan, A., Sadeghi, M., Ganjkhanlou, M., & Bahnamiri, H. Z. (2020). Reference gene selection in adipose and muscle tissues of fat-tailed Lori-bakhtiari lambs, Iranian Journal of Veterinary Medicine, 14(03), 273-288. doi: 10.22059/IJVM.2020.285371.1005008

Bradley, R. (1978). Skeletal muscle biopsy techniques in animals for histochemical and ultrastructural examination and especially for the diagnosis of myodegeneration. British Veterinary Journal, 134(5), 434-444. doi: 10.1016/S0007-1935(17)33385-7

Breuss, M., & Keays, D. A. (2014). Microtubules and neuro developmental disease: the movers and the makers. Advances in Expreimental Medicine and Biology, 800(1), 75-96. doi: 10.1007/978-94-007-7687-6

Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M.,… Shipley, G. L. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4), 611-622. doi: 10.1373/clinchem.2008.112797

Cafferky, J., Hamill, R. M., Allen, P., O Doherty, J. V., Cromie, A., & Sweeney, T. (2019). Effect of breed and gender on meat quality of M. longissimus thoracis et lumborum muscle from crossbred beef bulls and steers. Foods, 8(173), 1-10. doi: 10.3390/foods8050173

Clark, E. L., Bush, S. J., Mcculloch, M. E. B., Farquhar, I. L., Young, R., Lefevre, L.,… Hume, D. A. (2017). A high resolution atlas of gene expression in the domestic sheep (Ovis aries). PLoS Genetics, 13(9), e1006997.

Gagaoua, M., Terlouw, E. M. C., Micol, D., Hocquette, J., Moloney, A. P., & Nuernberg, K. (2016). Sensory quality of meat from eight different types of cattle in relation with their biochemical characteristics. Journal of Integrative Agriculture, 15(7), 1550-1563. doi: 10.1016/S2095-3119(16)61340-0

Garrels, J. I., & Gibsont, W. (1976). Identification and characterization of multiple forms of Actin. Cell, 9(4), 793-805. doi: 10.1016/0092-8674(76)90142-2

Gierke, S., Kumar, P., & Wittmann, T. (2010). Analysis of microtubule polymerization dynamics in live cells. Methods in cell biology, 97(10), 15-33. doi: 10.1016/S0091-679X(10)97002-7

Gong, H., Sun, L., Chen, B., Han, Y., Pang, J., Wu, W., & Qi, R. (2016). Evaluation of candidate reference genes for RT-qPCR studies in three metabolism related tissues of mice after caloric restriction. Scientific Reports, 6(1), 1-12. doi: 10.1038/srep38513

Guerrero, A., Valero, M. V., Campo, M. M., & Sañudo, C. (2013). Some factors that affect ruminant meat quality: from the farm to the fork. Review. Acta Scientiarum - Animal Sciences, 35(4), 335-347. doi: 10. 4025/actascianimsci.v35i4.21756

Hederstedt, L., & Rutberg, L. (1981). Succinate dehydrogenase-a comparative review. Microbiological Reviews, 45(4), 542-555. doi: 0146-0749/81/120542-14302.00/0

Heinz, C., Mberema, H., & Sparagano, O. A. E. (2017). Expression stability of reference genes in the skeletal muscles of beef cattle. African Journal of Biotechnology, 16(6), 261-267. doi: 10.5897/AJB2016.15140

Hernandez, D., Montes, D., Otero, R., Prieto, E., Espitia, A., & Buelvas, M. (2019). Polimorfismos FecXR, FecGI y FecGH en el ovino de pelo colombiano. Archivos de Zootecnia, 68(262), 194-200. doi: 10.21 071/az.v68i262.4136

Hildyard, J. C. W., Finch, A. M., & Wells, D. J. (2019). Identification of qPCR reference genes suitable for normalizing gene expression in the mdx mouse model of Duchenne muscular dystrophy. PLoS One, 14(1), 1-28. doi: 10.1371/ journal.pone.0211384

Hiller, B., Herdmann, A., & Nuernberg, K. (2011). Dietary n-3 fatty acids significantly suppress lipogenesis in bovine muscle and adipose tissue: a functional genomics approach. Lipids, 46(7), 557-567. doi: 10.10 07/s11745-011-3571-z

Holdridge (1987). Ecología basada en zonas de vida (No. 83). Agroamérica, 6. 199-203.

Huggett, J., Dheda, K., Bustin, S., & Zumla, A. (2005). Real-time RT-PCR normalisation; strategies and considerations. Genes and Inmmunity, 6(4), 279-284. doi: 10.1038/sj.gene.6364190

Jassim, A. I. (2014). Comparison of the anesthetic effect of paracetamol, tramadol, ketamine, xylazine, combination in three anesthetic protocols by using two different administration routes in sheep. Al-Qadisiyah Journal of Veterinary Medicine Sciences, 13(2), 137-145. doi: 10.29079/vol13iss2art315

Jiang, X. I. N., Xue, Y., Zhou, H., Li, S., & Zhang, Z. (2015). Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep. Molecular Medicine Reports, 12(4), 5633-5640. doi: 10.3892/mmr.2015.4128

Kawai, K., Miyazaki, S., Kawashima, T., Tajiri, E., Mori, M., Kitazaki, K.,… Yokoyama, M. (2010). Serum beta2-microglobulin concentration as a novel marker to distinguish levels of risk in acute heart. Journal of Cardiology, 55(1), 99-107. doi: 10.1016/j.jjcc.2009.10.003

Kim, J. W., Kim, S. J., Han, S. M., Paik, S. Y., Ph, D., Hur, S. Y.,… Namkoong, S. E. (1998). Increased glyceraldehyde-3-phosphate dehydrogenase gene expression in human cervical cancers. Gynecologic Oncology, 71(2), 266-269. doi: 10.1006/gyno.1998.5195

Kulichenko, A. N., Kovalev, D. A., Pisarenko, S. V, & Volynkina, A. S. (2016). Correlation between gene expression profiles in muscle and live weight in Dzhalginsky Merino sheep. Revista Colombiana de Ciencias Pecuarias, 29(3), 188-198. doi: 10.17533/udea.rccp.v29n3a04

Lind, V., Berg, J., Morten, S., Hersleth, M., & Olav, L. (2011). Effect of gender on meat quality in lamb from extensive and intensive grazing systems when slaughtered at the end of the growing season. Meat Sciencet, 88(2), 305-310. doi: 10.1016/j.meatsci.2011.01.008

Liu, J., & Victor, K. (2003). Estradiol and testosterone have opposite effects on microtubule polymerization. Neuro Endocrinology, 77(4), 258-272. doi: 10.1159/000070281

Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2- CT method. Methods, 25(4), 402-408. doi: 10.1006/meth.2001.1262

Mateescu, R. G., & Thonney, M. L. (2002). Gene expression in sexually dimorphic muscles in sheep 1. Journal of Animal Science, 80(7), 1879-1887. doi: 10.2527/2002.8071879x

Michal, J., Marketa, C., Jiri, R., Vojtech, S., Tetyana, S., Eduarda, D.,… David, S. (2018). Estradiol dimer inhibits tubulin polymerization and microtubule dynamics. Journal of Steroid Biochemistry and Molecular Biology, 183(1), 68-79. doi: 10.1016/j.jsbmb.2018.05.008

Moolchand, M., Kachiwal, A. B., Soomro, S. A., & Bhutto, Z. A. (2018). Comparison of sedative and analgesic effects of xylazine, detomidine, and medetomidine in sheep. Egyptian Journal of Sheep & Goat Sciences, 9(2), 43-48. doi: 10.21608/ejsgs.2014.26737

Mori, R., Wang, Q., Danenberg, K. D., Pinski, J. K., & Danenberg, P. V. (2008). Both B-Actin and GAPDH are useful reference genes for normalization of quantitative RT-PCR in human FFPE tissue samples of prostate cancer. The Prostate, 68(14), 1555-1560. doi: 10.1002/pros.20815

Nascimento, C. S., Barbosa, L. T., Brito, C., Fernandes, R. P. M., Mann, R. S., Pinto, A. P. G.,... Duarte, M. S. (2015). Identification of suitable reference genes for real time quantitative polymerase chain reaction assays on pectoralis major muscle in chicken (Gallus gallus). PLoS ONE, 10(5), 1-15. doi: 10.1371/ journal.pone.0127935

Nazari, F., Parham, A., & Maleki, A. F. (2015). GAPDH, beta-actin and beta 2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose and marrow-derived mesenchymal stem cells. Journal of Animal Science and Technology, 7(57), 1-8. doi: 10.1186/s40781-0 15-0050-8

Niu, G., Yang, Y., Zhang, Y., & Hua, C. (2016). Identifying suitable reference genes for gene expression analysis in developing skeletal muscle in pigs. PeerJ, 4(e2428), 1-19. doi: 10.7717/peerj.2428

Nolan, T., Hands, R. E., & Bustin, S. A. (2006). Quantification of mRNA using real-time RT-PCR. Nature Protocols, 1(3), 1559-1582. doi: 10.1038/nprot.2006.236

Peletto, S., Bertuzzi, S., Campanella, C., Modesto, P., Caramelli, M., & Acutis, P. L. (2011). Evaluation of internal reference genes for quantitative expression analysis by real-time PCR in ovine whole blood. International Journal of Molecular Sciences, 12(11), 7732-7747. doi: 10.3390/ijms12117732

Persons, D. A., Schek, N., Hall, B. L., & Finn, O. J. (1989). Increased expression of glycolysis-associated genes in oncogene-transformed and growth-accelerated states. Molecular Carcinogenesis, 94(2), 88-94.

Pfaffl, M. W., Tichopad, A., Prgomet, C., & Neuvians, T. P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnology Letters, 26(6), 509-515. doi: 10.1023/b:bile.0000019559.84305.47

Rapacz, M. (2013). Reference genes in real-time PCR. Journal of Applied Genetics, 54(4), 391-406. doi: 10.10 07/s13353-013-0173-x

Ruedrich, E. D., Henzel, M. K., Hausman, B. S., & Bogie, K. M. (2013). Reference gene identification for reverse transcription-quantitative polymerase chain reaction analysis in an ischemic wound-healing model. Journal of Biomolecular Techniques, 24(4), 181-186. doi: 10.7171/jbt.13-2404-003

Sahu, A. R., Wani, S. A., Saxena, S., Rajak, K. K., Malla, W. A., Ishaq, R.,… Mishra, B. (2018). Selection and validation of suitable reference genes for qPCR gene expression analysis in goats and sheep under Peste des petits ruminants virus (PPRV), lineage IV infection. Scientific Reports, 8(1), 1-11. doi: 10.10 38/s41598-018-34236-7

Schulze, F., Malhan, D., Khassawna, T. El, Heiss, C., Seckinger, A., Hose, D., & Rosen-Wolff, A. (2017). A tissue-based approach to selection of reference genes for quantitative real-time PCR in a sheep osteoporosis model. BMC Genomics, 18(975), 1-9. doi: 10.1186/s12864-017-4356-4

Sikand, K., Singh, J., Ebron, J. S., & Shukla, G. C. (2012). Housekeeping gene selection advisory: Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) and beta-actin are targets of miR-644a. PLoS ONE, 7(10), 1-9. doi: 10.1371/journal.pone.0047510

Staats, K. A., Schonefeldt, S., Van Rillaer, M., Van Hoecke, A., Van Damme, P., Robberecht, W.,… Van Den Bosch, L. (2013). Beta -2 microglobulin is important for disease progression in a murine model for amyotrophic lateral sclerosis. Frointiers in Cellular Neuroscience, 7(12), 6-10. doi: 10.3389/fncel. 2013. 00249

Tripathi, A. K., Koringa, P. G., Jakhesara, S. J., Ahir, V. B., Ramani, U. V., Bhatt, V. D.,… Joshi, C. G. (2012). A preliminary sketch of horn cancer transcriptome in Indian zebu cattle. Gene, 493(1), 124-131. doi: 10. 1016/j.gene.2011.11.007

Tristan, C., Shahani, N., Sedlak, T. W., & Sawa, A. (2011). The diverse functions of GAPDH : views from different subcellular compartments. Cellular Signalling, 23(2), 317-323. doi: 10.1016/j.cellsig.2010.08. 003

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2022-02-28

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Sandoval Lozano, E., & Rondon Barragan, I. S. (2022). Estabilidade de genes de referência no músculo Longissimus thoracis et lumborum de ovelhas crioulas colombianas. Semina: Ciências Agrárias, 43(3), 987–1006. https://doi.org/10.5433/1679-0359.2022v43n3p987

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