Selection of referent transcript for normalization of gene expression in cervical cytology samples

Real-time polymerase chain reaction is widely used in gene expression studies but this requires an appropriate referent gene for data normalization. So far, no gold standard is available and the selection has to be empirically validated. The aim of this study was to identify the most stable referent gene in exfoliated cervical cells with different degrees of cervical pathology. Seventy-five samples were used, 18 were from normal cervices, 18 from cervical intraepithelial neoplasia (CIN) 1, 17 from CIN 2, 17 from CIN 3, and 5 from squamous cell carcinoma. Using NormFinder, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found to be the most stably expressed referent gene with a stability value of 0.37 across all lesion grades. Followed by RPL4 (stability value of 0.77) and β-actin (0.77), large ribosomal protein P0 (1.01), and PKG1 (1.02). The results of expression stability by geNorm showed that normal cervices were more varied, with stability values ranging from 2.85 to 3.46, with β-actin performing slightly better than GAPDH (M: 2.85 vs. 2.98). For the CIN 1 to 3, GAPDH was determined to be the most stably expressed gene (M: 0.94 to 1.37). The next most stable gene expressed was PKG1 (M: 1.02 to 1.44). However, the sample size for squamous cell carcinoma was too small for justification. In conclusion, overall GAPDH is the most stable referent gene expressed across all cervical lesion grades and is most suitable for normalization in gene expression studies.