Identifying an appropriate endogenous control for real-time PCR of microRNAs in colorectal liver metastases: A comparison of 18s rRNA and small nuclear RNA RNU6B.

Selection of an appropriate housekeeping gene as an endogenous control is essential for obtaining reliable results in semi-quantitative real-time (RT-)PCR. However, the cellular composition varies according to the site of metastasis, resulting in differences among tissue compartments. Consequently, a suitable endogenous control for microRNA (miRNA) PCR may differ depending on the target organ, making individual evaluation of candidate controls necessary. In the present study, liver metastases were investigated, and RNU6B was compared with the well-established 18s ribosomal RNA (18s rRNA), commonly used in mRNA PCR, to determine which gene is more suitable as an endogenous control for miRNA analysis in colorectal liver metastases (CLM). A total of 79 patients with stage IV colorectal carcinoma who underwent liver resection were included. RNA, including miRNA, was extracted and analyzed using RT-PCR. The expression levels of RNU6B and 18s rRNA in CLM tissues and paired healthy liver tissues were assessed, and compared with the characteristics of a theoretically ideal endogenous control. The mean Cq values of RNU6B were significantly lower than those of 18s rRNA in both CLM tissues and corresponding healthy liver tissues (P<0.001). Pearson correlation analysis demonstrated a strong correlation between CLM and healthy liver tissues for RNU6B (correlation coefficient ϱ=0.807), whereas only a weak correlation was observed for 18s rRNA (ϱ=0.398). Furthermore, the slope and y-intercept of the regression line for RNU6B were closer to those of an ideal regression line than those for 18s rRNA. Clinical parameters, including sex, age, primary tumor localization and stage, and chronicity of liver metastases, did not significantly influence the expression of either RNU6B or 18s rRNA. Overall, RNU6B demonstrated greater conformity to the characteristics of an ideal endogenous control than 18s rRNA for miRNA PCR in CLM tissues. Based on these findings, RNU6B is recommended as an endogenous control for RT-PCR analysis of miRNAs in metastatic colorectal cancer.