Identification of optimal housekeeping genes for examination of gene expression in bovine corpus luteum |
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| Authors: | Robert Rekawiecki Joanna Rutkowska Jan Kotwica |
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| Affiliation: | 1. Department of Animal Science, National Chung Hsing University, Taichung, Taiwan;2. Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan;3. Department of Poultry Science, Texas A&M University, College Station, TX, USA;1. School of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland;2. School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland;3. Teagasc, Animal and Bioscience Research Centre, Animal & Grassland Research and Innovation Centre, Athenry, County Galway, Ireland;4. Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland;5. National Institute for Bioprocessing Research & Training, Blackrock, County Dublin, Ireland;1. Laboratorio de Biología Celular y Molecular Aplicada, Instituto de Ciencias Veterinarias del Litoral (ICiVet-Litoral), Universidad Nacional del Litoral (UNL) / Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Esperanza, Santa Fe, Argentina;2. Facultad de Ciencias Veterinarias del Litoral, Universidad Nacional del Litoral (UNL), Esperanza, Santa Fe, Argentina;1. Division of Cardiology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan;2. Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan;3. Division of Cardiovascular Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan;4. Department of Pathology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan;5. Department of Medicine, Faculty of Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan;6. Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan;2. Department of Obstetrics and Gynecology, Gynecological Endocrinology Center, University of Modena and Reggio Emilia;3. Department of Obstetrics and Gynecology, University of Pisa, Italy |
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| Abstract: | The selection of proper housekeeping genes for studies requiring genes expression normalization is an important step in the appropriate interpretation of results. The expression of housekeeping genes is regulated by many factors including age, gender, type of tissue or disease. The aim of the study was to identify optimal housekeeping genes in the corpus luteum obtained from cyclic or pregnant cows. The mRNA expression of thirteen housekeeping genes: C2orf29, SUZ12, TBP, TUBB2B, ZNF131, HPRT1, 18s RNA, GAPDH, SF3A1, SDHA, MRPL12, B2M and ACTB was measured by Real-time PCR. Range of cycle threshold (Ct) values of the tested genes varied between 12 and 30 cycles, and 18s RNA had the highest coefficient of variation, whereas C2orf29 had the smallest coefficient. GeNorm software demonstrated C2orf29 and TBP as the most stable and 18s RNA and B2M as the most unstable housekeeping genes. Using the proposed cut-off value (0.15), no more than two of the best GeNorm housekeeping genes are proposed to be used in studies requiring gene expression normalization. NormFinder software demonstrated C2orf29 and SUZ12 as the best and 18s RNA and B2M as the worst housekeeping genes. The study indicates that selection of housekeeping genes may essentially affect the quality of the gene expression results. |
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