Identification of reference genes and miRNAs for qRT-PCR in human esophageal squamous cell carcinoma.

It is important to select an appropriate reference gene and miRNA when using quantitative real-time polymerase chain reaction (qRT-PCR) to analyze gene and miRNA expression. However, many commonly used reference genes and miRNAs are not stably expressed and therefore not suitable for normalization or quantification of qRT-PCR data. This study aims to identify appropriate reference genes and miRNAs for use in human esophageal squamous carcinoma qRT-PCR analysis. Using data provided by The Cancer Genome Atlas, we identified DDX5, LAPTM4A, P4HB, RHOA, miR-28-5p, miR-34a-5p, and miR-186-5p as candidate reference genes and miRNAs. We used qRT-PCR to verify the expression levels of these candidates and another seven commonly used reference genes and miRNAs. A set of 50 paired human normal esophageal tissues and squamous cell carcinoma samples were used in the analysis. We then used geNorm and NormFinder to analyze the results. DDX5, LAPTM4A, RHOA, ACTB, RNU48, miR-28-5p, miR-34a-5p, and miR-186-5p were stably expressed, indicating they are suitable for used as references in qRT-PCR analysis of esophageal squamous cell carcinoma. However, expression levels of 18s rRNA, GAPDH, P4HB, 5s rRNA, U6, and RNU6B varied greatly between esophageal normal and squamous cell carcinoma samples, indicating that they are not suitable for use as references in the qRT-PCR analysis of esophageal squamous cell carcinoma.