Direct Assay of miR-223, miR-145, and miR-16 by a Novel Method of Mimic PCR in Serum of Breast Cancer Patients

The role of microRNAs (miRs) is shown as a biomarker whose expression level changes have been associated with cancer development and progression. Circulating miRNAs have been postulated as biomarkers for breast cancer. Detection and diagnosis of miRNA markers could provide an improved and sensitive method in the clinical application. In this study, we applied and improved the effective approach by coupling a deoxyuridine-incorporated RT oligonucleotide with a secondary structure and a mimic PCR for assessment and direct detection of circulating miRs in serum. We designed mut miR-16, mut miR-145, and mut miR-223 as mimic miRs. These mimic miRs were used in RT-PCR for detection and quantification assay. As normalization of these mut miRs was performed with themselves, our mimic construct with 80 bp of these miRs shows the best normalization for this method in blood samples. This assay was tested in serum from 15 patients with different stages of breast cancer and 10 healthy female donors. According to our results, using a combination of miR-16 and miR-145 could represent as one of the best biomarker (p