Reproducible and Reliable Real-time PCR Assay to Measure Mature Form of miR-141.

Abstract

miR-141 is one of the miRNAs that has significant expression variations in different human malignancies including prostate cancer, hepatocellular carcinoma, renal cell carcinoma, pancreatic cancer, gastric cancer, and ovarian cancer. Furthermore, various studies have designated miR-141 as a prognostic and diagnostic biomarker in different types of cancer. Thus, accurate and precise quantification of miR-141 is very essential for clinical diagnostics. In this regard, development of a reproducible and reliable assay for miR-141 can be the first step to standardize quantification of this valuable biomarker for in vitro diagnostics assays. Using stem-loop approach, we designed a Taqman real-time PCR assay for miR-141. This method allowed us to reproducibly and reliably quantify miR-141. The specificity, sensitivity, interassay and intraassay, and the dynamic range of the method were determined. The assay had a linear dynamic range of 3E-9.6E copies/reaction and the limit of detection was determined to be between 960 and 192 copies/reaction with 95% confidence interval. In addition, the R2 rate was >0.99 and the slope of the standard curve >-3.27, indicating great amplification efficiency, which is >99%. The coefficient of variation for Ct values was <1.9% and 2.39% for intraassay and interassay, respectively. Therefore, this study can be the first step to standardize miR-141 evaluations, which consequently assist the physicians for improved prognosis, diagnosis, and treatment.