Volume: 57 Issue: 3
Year: 2026, Page: 475-480, Doi: https://doi.org/10.51966/jvas.2026.57.3.475-480
Received: July 9, 2026 Accepted: Sept. 11, 2026 Published: Sept. 30, 2026
Accurate normalisation of quantitative real-time PCR (qPCR) data depends critically on the use of a stably expressed reference gene. Hence reference genes traditionally assumed to be constitutively expressed can show considerable tissue- and condition-specific variability. In the present study, the expression stability of three commonly used candidate reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB) and TATA box binding protein (TBP), was evaluated in ten samples each from Psaos major and Biceps femoris muscles of cattle, with a view to identifying the most suitable reference gene for normalising the expression of tenderness-related target genes in cattle. Quantification cycle (CT ) values for the three candidate genes were obtained from 20 muscle samples by qPCR and analysed using four independent approaches: geNorm, NormFinder, BestKeeper and the comparative ΔCT method. All four methods consistently ranked GAPDH as the most stably expressed of the three candidate genes, followed by ACTB, with TBP identified as the least stable. On the basis of this consensus ranking, GAPDH was selected as the reference gene for subsequent qPCR-based quantification of tenderness-related gene expression.
Keywords: Reference gene, GAPDH, geNorm, normfinder, bestkeeper, comparative ΔCT method
Andersen, C. L., Jensen, J. L., & Orntoft, T. F. (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 Research, 64(15), 5245–5250. https://doi.org/10.1158/0008-5472.CAN-04-0496
Freitas, F. C. P., Depintor, T. S., Agostini, L. T., Luna-Lucena, D., Nunes, F. M. F., Bitondi, M. M. G., Simões, Z. L. P., & Lourenço, A. P. (2019). Evaluation of reference genes for gene expression analysis by real-time quantitative PCR (qPCR) in three stingless bee species (Hymenoptera: Apidae: Meliponini). Scientific Reports, 9, Article 17692. https://doi. org/10.1038/s41598-019-53544-0
Kumar, S., Muthukumar, M., Bajpai, A., Kushwaha, A. K., Ahmad, I., Bajpai, Y., Singh, A., Damodaran, T., & Trivedi, M. (2025). Selection and validation of stable reference genes in guava (Psidium guajava L.) for reliable and consistent gene expression analysis. Electronic Journal of Biotechnology, 75, 49–56. https://doi.org/10.1016/j.ejbt.2025.01.006
Ma, J., Chen, J., Gan, M., Chen, L., Zhao, Y., Niu, L., Zhu, Y., Zhang, S., Li, X., Guo, Z., Wang, J., Zhu, L., & Shen, L. (2022). Comparison of reference gene expression stability in mouse skeletal muscle via five algorithms. PeerJ, 10, Article e14221. https:// doi.org/10.7717/peerj.14221 Nygard, A. B., Jørgensen, C. B., Cirera, S., & Fredholm, M. (2007). Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Molecular Biology, 8, Article 67. https:// doi.org/10.1186/1471-2199-8-67
Panina, Y., Germond, A., Masui, S., & Watanabe, T. M. (2018). Validation of common housekeeping genes as reference for qPCR gene expression analysis during iPS reprogramming process. Scientific Reports, 8, Article 8716. https://doi.org/10.1038/ s41598-018-26707-8
Pérez, R., Tupac-Yupanqui, I., & Dunner, S. (2008). Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue. BMC Molecular Biology, 9, Article 79. https://doi. org/10.1186/1471-2199-9-79
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. https://doi.org/10.1023/ B:BILE.0000019559.84305.47
Sahu, A. R., Wani, S. A., Saxena, S., Rajak, K. K., Chaudhary, D., Sahoo, A. P., Khanduri, A., Pandey, A., Mondal, P., Malla, W. A., Khan, R. I. N., Tiwari, A. K., Mishra, B., Muthuchelvan, D., Mishra, B. P., Singh, R. K., & Gandham, R. K. (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, Article 15969. https://doi.org/10.1038/s41598-018-34236-7.
Silver, N., Best, S., Jiang, J., & Thein, S. L. (2006). Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Molecular Biology, 7, Article 33. https://doi. org/10.1186/1471-2199-7-33
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 Biology, 3(7), Article research0034. https://doi.org/10.1186/gb 2002-3-7-research0034
Wang, G. H., Liang, C. C., Li, B. Z., Du, X. Z., Zhang, W. Z., Cheng, G., & Zan, L. S. (2022). Screening and validation of reference genes for qRT-PCR of bovine skeletal muscle-derived satellite cells. Scientific Reports, 12, Article 5653. https://doi.org/10.1038/ s41598-022-09476-3
Zhu, W., Lin, Y., Liao, H., & Wang, Y. (2015). Selection of reference genes for gene expression studies related to intramuscular fat deposition in Capra hircus skeletal muscle. PLOS ONE, 10(3), Article e0121280. https://doi.org/10.1371/journal. pone.0121280
© 2026 Aswani et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Raj B. A., Radhakrishnan U., Muhammed S., John, L., Vasudevan, V. N., Panicker, V. P., Chinnu M. V. & Binu, S. (2026). Validation of reference gene stability for normalisation of qPCR in bovine skeletal muscles. Journal of Veterinary and Animal Sciences, 57(3), 475-480