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<p>Intestinal organoids (IOs) have been efficiently used in studies on intestinal epithelial homeostasis and disorders. However, the reliable quantification of their gene expression remains a challenge because of their intrinsic complexity and heterogeneity, along with the intrinsic features of the intestinal tissue as the fastest proliferating mammalian tissue. Even though quantitative reverse transcription polymerase chain reaction (RT-qPCR) is widely used for genotypic analysis of organoids, the selection of appropriate housekeeping genes for data normalization remains critical, given the inherent batch-to-batch variations in current organoid cultures. Here, to identify and validate the most stable housekeeping genes for IO analysis, we systematically analyzed the expression stability of nine housekeeping genes (GAPDH, ACTB, 18S, SDHA, B2M, RPL4, YWHAZ, PPIA, and HPRT1) in both intestinal tissue and IO samples using four established statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Our analysis revealed that SDHA and ACTB exhibited the highest stability across all the algorithms; conversely, YWHAZ showed suboptimal stability in our experimental context. Validation experiments using epithelial gland cell markers (Lgr5 and Muc2) confirmed that normalization with stable housekeeping genes (SDHA and ACTB) leads to consistency on target gene expression, while less stable gene (YWHAZ) gives rise to significantly different outcomes between intestinal tissues and IOs. Our findings demonstrate that the careful selection of housekeeping genes remarkably influences the accuracy and reliability of the gene expression analysis of IOs. This study can serve as a basis for establishing more standardized and reliable gene expression analyses in translational sciences.</p>

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Identification and validation of robust housekeeping genes for quantitative genotypic analyses in translational intestinal organoid studies

  • 期刊论文

  • 发表日期：

    2025年7月23日

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  • Dong Chang Kim,
  • Sung-Ho Kim,
  • Min Beom Kim,
  • Won-Jae Lee,
  • Yun Kee Jo

摘要

Intestinal organoids (IOs) have been efficiently used in studies on intestinal epithelial homeostasis and disorders. However, the reliable quantification of their gene expression remains a challenge because of their intrinsic complexity and heterogeneity, along with the intrinsic features of the intestinal tissue as the fastest proliferating mammalian tissue. Even though quantitative reverse transcription polymerase chain reaction (RT-qPCR) is widely used for genotypic analysis of organoids, the selection of appropriate housekeeping genes for data normalization remains critical, given the inherent batch-to-batch variations in current organoid cultures. Here, to identify and validate the most stable housekeeping genes for IO analysis, we systematically analyzed the expression stability of nine housekeeping genes (GAPDH, ACTB, 18S, SDHA, B2M, RPL4, YWHAZ, PPIA, and HPRT1) in both intestinal tissue and IO samples using four established statistical algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Our analysis revealed that SDHA and ACTB exhibited the highest stability across all the algorithms; conversely, YWHAZ showed suboptimal stability in our experimental context. Validation experiments using epithelial gland cell markers (Lgr5 and Muc2) confirmed that normalization with stable housekeeping genes (SDHA and ACTB) leads to consistency on target gene expression, while less stable gene (YWHAZ) gives rise to significantly different outcomes between intestinal tissues and IOs. Our findings demonstrate that the careful selection of housekeeping genes remarkably influences the accuracy and reliability of the gene expression analysis of IOs. This study can serve as a basis for establishing more standardized and reliable gene expression analyses in translational sciences.