Inability to compare β-actin expression with RT-qPCR in bovine liver samples kept under different conditions

Gene expression analyses play a critical role in understanding cellular processes and elucidating the molecular basis of diseases. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR), a highly sensitive method, is widely used for measuring gene expression levels. However, the success of such analyses is largely dependent on the integrity of the biological samples used. In particular, the storage and fixation conditions of tissue samples, especially those preserved for long-term or archival purposes, directly affect RNA quality. In this study, lung tissues obtained from a healthy bovine subject were prepared under six different conditions—fresh, frozen, autolyzed, formalin-fixed, formalin-fixed paraffin-embedded (FFPE), and alcohol-fixed—to evaluate the impact of storage conditions on gene expression reliability. Expression levels of the β-actin gene were analyzed using the RT-qPCR technique. The findings revealed that RNA quality and amplification efficiency differed significantly depending on the preservation method. The highest RNA integrity and gene expression efficiency were observed in fresh and frozen tissues, while markedly lower expression levels were detected in FFPE and autolyzed samples. These results indicate that appropriate preservation methods are crucial for obtaining reliable gene expression data, particularly in studies involving archived tissue materials. Keywords: β-actin, liver, RT-qPCR, bovine