Validation of reference genes for gene expression studies in bovine oocytes and cumulus cells derived from in vitro maturation
Lisandra Cristina Caetano, Cristiana Libardi Miranda-Furtado, Luciene Aparecida Batista, Caroline Palmieri Pitangui-Molina, Thaís Tiemi Higa, Cristiana Carolina Padovan, Ana Carolina Japur de Sá Rosa-e-Silva
Abstract
Quantitative real-time PCR (qPCR) is a valuable tool for gene expression studies and it is necessary to choose an ideal endogenous reference gene for data normalization. This work studied a set of reference genes in oocytes and cumulus cells of COCs (Cumulus-Oocyte Complexes) that are suitable for relative gene expression analyses after in vitro maturation (IVM) in bovine. Immature COCs were collected from ovaries of Nelore cattle (Bos indicus) and submitted to IVM. MII oocytes and cumulus cells were subjected to RNA extraction, reverse transcription and preamplification of cDNA. The expression level of eight reference genes (ACTB, GADPH, B2M, H2AFZ, GUSB, HPRT1, PPIA, and TBP) was measured by real time PCR and analyzed by geNorm software. The gene stability measure (M) was calculated and the ideal number of reference genes (RGs) was determined by the V value (pairwise variation). For oocyte samples, two RGs were the ideal number for relative quantification: HPRT1 and B2M and for bovine cumulus samples four were indicated: HPRT1, PPIA, B2M, and TBP genes. The normalization of a non-reference target gene (SOD1) by these reference genes was shown to be considerably different from normalization by less stable reference genes. Our results strengthen the importance of choosing good normalizing genes in order to analyze gene expression under specific experimental conditions and we suggest the use of these RGs in oocytes and cumulus cells of bovine cattle in in vitro matured COCs.
Keywords
References
Adriaenssens T, Wathlet S, Segers I, Verheyen G, De Vos A, Van der Elst J, Coucke W, Devroey P, Smitz J. 2010. Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics. Hum Reprod, 25:1259-1270.
Andersen CL, Jensen JL, Ørntoft TF. 2004. 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, 64:5245-5250.
Biase FH, Martelli L, Merighe GK, Santos Biase WK, Miranda M, Smith LC, Meirelles FV. 2009. A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome. Zygote, 17: 289-295.
Bruzzone R, White TW, Paul DL. 1996. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem, 238:1-27.
Cammack KM. 2009. Reproductive Traits and Their Heritabilities in Beef Cattle. The Professional Animal Scientis, 25:517-528.
Cetica PD, Pintos LN, Dalvit GC, Beconi MT. 2001. Antioxidant enzyme activity and oxidative stress in bovine oocyte in vitro maturation. IUBMB Life, 51:57-64.
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, 344:141-143.
Gilchrist RB, Lane M, Thompson JG. 2008. Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Hum Reprod Update, 14:159-177.
Gosden RG. 2002. Oogenesis as a foundation for embryogenesis. Mol Cell Endocrinol, 186:149-153.
Hamel M, Dufort I, Robert C, Gravel C, Leveille MC, Leader A, Sirard MA. 2008. Identification of differentially expressed markers in human follicular cells associated with competent oocytes. Hum Reprod, 23:1118-1127.
Hruz T, Wyss M, Docquier M, Pfaffl MW, Masanetz S, Borghi L, Verbrugghe P, Kalaydjieva L, Bleuler S, Laule O, Descombes P, Gruissem W, Zimmermann P. 2011. RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization. BMC Genomics, 12:156.
Khan MI, Dias FC, Dufort I, Misra V, Sirard MA, Singh J. 2016. Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging, Reprod Fertil Dev, 28:795-805.
Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25:402-408.
Macabelli CH, Ferreira RM, Gimenes LU, de Carvalho NA, Soares JG, Ayres H, Ferraz ML, Watanabe YF, Watanabe OY, Sangalli JR, Smith LC, Baruselli PS, Meirelles FV, Chiaratti MR. 2014. Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer. PLoS One. 9:e93287.
Marquant-Le Guienne B. 1999. Atlas of the bovine oocyte AETE Newsletter, 10:6-8.
Patel OV, Bettegowda A, Ireland JJ, Coussens PM, Lonergan P, Smith GW. 2007. Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes. Reproduction, 133:95-106.
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. Biotechnology Letters, 26:509-515.
Radonić 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.
Ragni E, Viganò M, Rebulla P, Giordano R, Lazzari L. 2013. What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes. J Cell Mol Med, 17:168-180.
Ross DG, Bowles J, Hope M, Lehnert S, Koopman P. 2009. Profiles of gonadal gene expression in the developing bovine embryo. Sex Dev, 3:273-283.
Salhab M, Dhorne-Pollet S, Auclair S, Guyader-Joly C, Brisard D, Dalbies-Tran R, Dupont J, Ponsart C, Mermillod P, Uzbekova S. 2013. In vitro maturation of oocytes alters gene expression and signaling pathways in bovine cumulus cells. Mol Reprod Dev, 80:166-182.
Senbon S, Hirao Y, Miyano T. 2003. Interactions between the oocyte and surrounding somatic cells in follicular development: lessons from in vitro culture. J Reprod Dev, 49:259-269.
Sugiura K, Pendola FL, Eppig JJ. 2005. Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism. Dev Biol, 279:20-30.
Sutton ML, Gilchrist RB, Thompson JG. 2003. Effects of in-vivo and in-vitro environments on the metabolism of the cumulus-oocyte complex and its influence on oocyte developmental capacity. Hum Reprod Update, 9:35-48.
Vandesompele 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(7):RESEARCH0034.
Facebook
Google+
Twitter
LinkedIn
Mendeley
StumbleUpon
CiteULike
Reddit
Email