Identification of suitable reference genes for measurement of gene expression in human cervical tissues

Abstract

For quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), the most commonly used normalization strategy is to select a stable reference gene. However, no suitable reference genes have been identified in cervical tissues to date. The aim of this study was to identify the most stable gene or a set of genes as reference genes for RT-qPCR analysis in cervical tissues from a panel of 12 candidates (ALAS1, PPIA, GAPDH, HBB, TBP, ACTIN, B2M, MBNL2, PGKL, RPLP0, RPL-4, and EEF1A1). In total, 20 normal and 20 cervical cancer specimens were examined. Gene expression data were analyzed using two different statistical models (geNorm and NormFinder). EEF1A1 was identified as the most stable and reliable reference gene, followed by GAPDH and RPLP0, whereas EEF1A1 and GAPDH were the best two-gene combination by NormFinder. The expression validity of EEF1A1 was further determined in 21 normal, 22 cervical intraepithelial neoplasia (CIN(2-3)), and 18 cancer tissues; no expression differences were found among normal, CIN(2-3), and cancer tissues (P>0.05). Our results suggested that EEF1A1 can be used as a reference gene for normalization in gene profiling studies in clinic cervical samples, and the combination of EEF1A1 and GAPDH could be recommended as a much more reliable normalization strategy.