Geometric Mean of 5S rRNA and MiR-16 As A Suitable Normalizer In Esophageal Cancer

Abstract

Esophageal squamous cell carcinoma (ESCC) is a deadly cancer with poor prognosis. In this regard, early diagnosis is of vital importance to cure the tumor in its early stages. Novel cancer diagnostic and therapeutic approaches have been recently introduced based on microRNAs (miRNAs). Also, accurate normalization using appropriate reference genes is a critical step in miRNA expression studies. In this study, we aimed to identify appropriate reference genes for miRNA quantification in serum samples of ESCC. In this case and control experimental study, two statistical algorithms including GeNorm and NormFinder were used to evaluate the suitability of miR-16 and 5S rRNA and their geometric mean as reference genes. Then, relative expression of miR-451 and miR-24 were evaluated while different normalizer including miR-16, 5S rRNA and their geometric mean were applied. Both GeNorm and NormFinder analyses showed that geometric mean of miR-16 and 5S rRNA is the most stable reference gene in these samples. Also, our data showed that choosing an inappropriate normalizer could change the relative expression of target genes of miR-451 and miR-24 in ESCC samples which emphasize on the importance of selecting a reliable internal control in expression analyses. We demonstrated that geometric mean of two reference genes could increase the reliability of normalizers and also by using geometric mean as reference gene, relative expression of different target is closer to reality.