Identification of reference genes specific to parathyroid and thyroid tissues in rats

Gene expression analysis stands as one of the most powerful and fundamental tools in molecular biology and biotechnology to unravel the functional activity of genes at the transcriptional level. Among the various methods, quantitative real-time Polymerase Chain Reaction (qRT-PCR) is highly favored for its exceptional sensitivity, speed, and reliability in measuring relative gene expression levels. However, the accuracy of qRT-PCR results hinges critically on the appropriate selection and stability of reference genes, also known as housekeeping genes, which are used to normalize expression data. An ideal reference gene must maintain a constant expression level across different experimental groups, tissue types, and physiological conditions. The use of an unsuitable reference gene can introduce significant variability, leading to the misinterpretation of gene expression data and, ultimately, to incorrect biological conclusions. The primary objective of this thesis was to identify the most suitable reference genes for the reliable normalization of thyroglobulin (TG) and parathyroid hormone (PTH) expression, which are specific molecular markers for thyroid and parathyroid tissues, respectively. The study utilized both male and female individuals from two different rat strains, Sprague-Dawley and Wistar, to investigate potential variations in reference gene stability based on sex and strain. Nine candidate reference genes— RPLP13A, PGK1, SDHA, TBP, HPRT1, GAPDH, YWHAZ, VCL, and ACTB —were chosen for a comprehensive stability analysis. The expression stability of these genes was evaluated using a multi-algorithmic approach, integrating data from four widely recognized bioinformatics tools: BestKeeper, NormFinder, GeNorm, and the Delta Ct method. This robust strategy allowed for a detailed assessment of each gene's performance under various experimental conditions. The findings unequivocally demonstrated that reference gene stability is not a universal characteristic. Instead, it varies significantly depending on the biological variables examined, namely sex and strain. The comprehensive ranking analysis performed with the RefFinder tool provided a clear picture of these differences. Among the nine selected candidate genes, Ct values greater than 38 for the PGK1, HPRT1, YWHAZ, and VCL genes led to their exclusion from the stability analysis. In male rats, ACTB (geometric mean: 1.68) and SDHA (geometric mean: 1.86) were identified as the most stable reference genes, while in female rats, SDHA (geometric mean: 1.19) and TBP (geometric mean: 2.45) exhibited a superior stability profile. This sex-specific difference, particularly the notable decrease in ACTB's stability in females compared to males, underscores the crucial influence of sex on gene expression levels and highlights the necessity of considering this factor during the normalization process. Strain-specific analyses revealed similar variations. In Sprague-Dawley rats, TBP and ACTB genes demonstrated the highest stability, whereas in Wistar rats, SDHA and TBP genes were found to be more stable. This disparity highlights the importance of validating reference genes on a strain-by-strain basis to ensure the reliability of experimental outcomes. Furthermore, a highly significant finding was that GAPDH, a gene traditionally considered a universal housekeeping gene, displayed the lowest stability values across all groups and analytical methods examined in this study. This result challenges the conventional assumption of its suitability and provides strong evidence that the unvalidated use of GAPDH as a reference gene can lead to flawed or incomplete interpretations of results. In contrast, SDHA and TBP, which are involved in fundamental cellular processes such as the Krebs cycle and transcription initiation, generally exhibited high stability and consistency throughout the study. This research, therefore, provides a strong argument for the indispensable role of reference gene validation in qRT-PCR-based gene expression analyses. The results suggest that for studies involving thyroid and parathyroid tissues in Sprague-Dawley and Wistar rats, SDHA and TBP should be considered the primary choices for normalization. For optimal reliability, it is imperative that researchers select reference gene combinations tailored to their specific biological groups and experimental conditions. For instance, the study recommends using ACTB and SDHA for male rats, and SDHA and TBP for female rats. The automatic use of GAPDH without prior validation is strongly discouraged in these and similar experimental models due to its observed instability. To maximize the accuracy and reproducibility of gene expression analyses, the thesis recommends a multi-algorithmic approach, as employed in this study. Relying on a single algorithm may not provide a complete picture of gene stability. Instead, a comparative evaluation of results from multiple tools like BestKeeper, NormFinder, and GeNorm is crucial for establishing a truly robust and reliable set of reference genes. In summary, this thesis demonstrates that the selection of an appropriate reference gene is a decisive factor in the integrity of qRT-PCR-based gene expression analysis. The comprehensive, multi-algorithmic evaluation performed here identified SDHA and TBP as the most stable and reliable genes for normalization in the context of TG and PTH expression in rat thyroid and parathyroid tissues. The framework provided by this study contributes to more robust molecular profiling in thyroid and parathyroid research and serves as a valuable guide for future investigations into tissue-specific gene expression using real-time PCR. This work's emphasis on the necessity of experimental validation for reference genes underscores its importance in ensuring the scientific rigor and reproducibility of gene expression studies.