Assessment of endogenous reference gene suitability for serum exosomal microRNA expression analysis in liver carcinoma resection studies

Serum exosomal microRNAs (miRNAs) have received considerable attention as potential biomarkers for tumor diagnosis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is commonly used to detect miRNA expression levels in various types of cancer. One prerequisite for valid RT-qPCR data is the correct normalization of miRNAs to stably expressed endogenous reference genes (RGs). The study of liver carcinoma resection requires the use of reliable RGs in order to assess the expression levels of serum exosomal target miRNAs. However, the assessment of RG suitability for optimum serum exosomal miRNA expression analysis has yet to be investigated. The present study investigated the expression stability of 10 candidate RGs. The candidate genes included eight miRNAs (miR-16, miR-103, miR-191, let-7a, miR-26a, miR-221, miR-181a, and miR-451) and two small RNAs (5S and U6). The stability values of the candidate genes were calculated using the following algorithms: geNorm, NormFinder, BestKeeper, and the comparative Delta Ct method. The overall ranking obtained from these analyses revealed that miR-221, let-7a, and miR-26a were appropriate internal RGs for analysis of serum miRNAs in patients with hepatocellular carcinoma. In addition, normalization with miR-221 and let-7a combined, as recommended by geNorm, or with miR-26a, as recommended by NormFinder, increased the accuracy of interpretation of the target miRNA expression levels in hepatopathy studies.