Validation of Appropriate Reference Genes for Gene Expression Studies in Human Thyroid Gland Using Real-Time RT-PCR
Suchart Chantawibul MD*, Angkoon Anuwong MD*, Kawin Leelawat MD*
Affiliation : * Department of Surgery, Rajavithi Hospital, College of Medicine, Rangsit University Bangkok, Thailand
Background : Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription
PCR (qRT-PCR) becoming the method of choice. The selection of reference genes is critical for gene expression studies
because the expression of these genes may vary among tissues or cells and may change under certain circumstances.
However, there has not been any study that compares the stability of these reference genes in human thyroid specimens.
Therefore, the authors studied the stability values and the appropriate reference genes expressed in thyroid specimens.
Material and Method: 25 human thyroid specimens were prospectively collected and extracted for their RNA. The candidate
reference genes (hypoxanthine phosphoribosyl-transferase1 (HPRT1), ribosomal protein LI3a (RPLIA), β-2-micro-globulin
(B2M), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPD)) were amplified from these thyroid specimens
using real-time RT-PCR. The stabilities of these candidate reference genes were analyzed using Normfinder® software.
Results : The authors found that HPRT1 has the highest stability value (40.38 x 109) while GAPD has the lowest stability value
(85.46 x 107). Therefore GAPD is the most stably expressed gene in thyroid specimens.
Conclusion : Of the 5 genes studied, GAPD was found to be the best reference gene for gene expression studies in the thyroid
gland. The present results may facilitate the choice of reference genes for expression studies in thyroid diseases.
Keywords : Gene expression, Housekeeping gene, RT-PCR, Thyroid diseases, Normfinder
