Validation of reference genes for normalization gene expression in reverse transcription quantitative PCR in human normal thyroid and goiter tissue.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been recognized as the most accurate method for quantifying mRNA transcripts, but normalization of samples is a prerequisite for correct data interpretation. So, this study aimed to evaluate the most stable reference gene for RT-qPCR in human normal thyroid and goiter tissues. Beta-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); succinate dehydrogenase, subunit A, flavoprotein (Fp) (SDHA); hypoxanthine phosphoribosyltransferase I (HPRTI); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ); and beta-2-microglobulin (B2M) were evaluated in 14 thyroid tissue samples (7 normal and 7 goiter tissues) by RT-qPCR. The mean Cq and the maximum fold change (MFC) and NormFinder software were used to assess the stability of the genes. As a result, ACTB gene was more stable than GAPDH, SDHA, HPRTI, YWHAZ, and B2M. In conclusion, ACTB could be used to normalize RT-qPCR data in normal thyroid and goiter tissues.

Validation of reference genes for normalizing gene expression in real-time quantitative reverse transcription PCR in human thyroid cells in primary culture treated with progesterone and estradiol.

The use of appropriately chosen reference genes for normalizing gene expression in real-time quantitative reverse transcription polymerase chain reaction is an important step in the analysis of gene expression, compensating for several technical factors. As female sex hormones have been shown to influence growth and differentiation of thyroid follicular cells, the establishment of normalizer genes in human thyroid cells in primary culture, treated with progesterone, and estradiol, is important to evaluate their effect on gene expression in these cells, so candidate reference genes were studied. ß-Actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß2-microglobulin (B2M), and TATA box binding protein (TBP) were evaluated in thyroid cells treated with estradiol, progesterone, and their inhibitors. Normfinder software was used to assess the stability of the genes and identified ß-actin as the gene with adequate stability and lower inter-group variations, when compared to TBP, B2M, and GAPDH.