Evaluation of reference genes suitable for studying mRNAs and microRNAs expression in thyroid neoplasms

Analysis of gene expression is a pivotal method at the core of biomarkers studies and cancer research. Currently, RT-qPCR is the most commonly used technique to investigate the expression of certain genes. The accurate and reliable result relies on an effective normalization step using suitable reference genes. The present study was designed to evaluate the eligibility of a set of candidate mRNAs and snoRNA as reference genes in the most common human thyroid neoplasms. We tested the expression levels of eleven mRNA and small RNA housekeeping genes in thyroid samples. The stability of the candidate genes was examined in different thyroid lesions and under different experimental conditions. Results were compared to the reported data in the TCGA database. Our results suggested HPRT1 and ACTB as the best mRNA reference genes, SNORD96A, and SNORD95 as the best miRNA reference genes in thyroid tissues. These genes showed the most stable expression pattern among different thyroid lesions as well as different experimental conditions. The findings in this study highlight the effect of reference genes selection on data interpretation and emphasize the importance of testing for suitable reference genes to be used in specific types of cells and experimental conditions to ensure the validity and accuracy of results.