Identification of candidate reference genes for gene expression studies in buffalo cumulus cells
DOI:
https://doi.org/10.14295/bjs.v6i1.887Keywords:
cumulus cells, qPCR normalization, reference genes, buffaloAbstract
Quantitative PCR (qPCR) is a powerful technique for gene expression analysis; however, accurate data normalization requires the selection of appropriate endogenous reference genes. This study evaluated a panel of reference genes in cumulus cells derived from buffalo cumulus-oocyte complexes (COCs). In the present study, immature COCs were collected from the ovaries of water buffalo (Bubalus bubalis) and cultured in vitro. Cumulus cells from three independent biological replicates were used for total RNA isolation and cDNA synthesis. The expression stability of nine reference genes (GAPDH, B2M, β-ACTIN, PPIA, RPL15, H2AFZ, HPRT1, TBP, and GUSB) was assessed using qPCR. Gene stability was evaluated using Ref Finder, a comprehensive web-based tool for the comparative assessment of housekeeping genes. The expression stability of nine reference genes in cumulus cells was systematically evaluated using the RefFinder tool, which integrates geNorm, NormFinder, BestKeeper, and the comparative ΔCt method. The comprehensive stability ranking identified GUSB and GAPDH as the most stable reference genes across all samples. PPIA, RPL15, and HPRT exhibited moderate stability, whereas commonly used housekeeping genes, including TBP, β-ACTIN, and B2M, showed significant variability (P < 0.05) and were ranked among the least stable internal control genes for accurately normalizing gene expression in buffalo cumulus cells under the present experimental conditions.
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