title: A home-brew real-time PCR assay for reliable detection and quantification of mature miR-122.
abstract: miR-122 is a liver-specific miRNA that has significant gene expression alterations in response to specific pathophysiological circumstances of liver such as drug-induced liver injury, hepatocellular carcinoma, and hepatitis B and C virus infections. Therefore, accurate and precise quantification of miR-122 is very important for clinical diagnostics. However, because of the lack of in vitro diagnostics assays for miR-122 detection and quantification of the existence of an open-source assay could inevitably provide external evaluation by other researchers and the chance of promoting the assay when required. The aim of this study was to develop a Taqman real-time polymerase chain reaction assay, which is capable of robust and reliable quantification of miR-122 in different sample types. We used stem loop methodology to design a specific Taqman real-time polymerase chain reaction assay for miR-122. This technique enabled us to reliably and reproducibly quantify short-length oligonucleotides such as miR-122. The specificity, sensitivity, interassay and intra-assay, and the dynamic range of the assay were experimentally determined by their respective methodology. The assay had a linear dynamic range of 3E to 4.8E miR-122 copies/reaction and the limit of detection was determined to be between 960 and 192 copies/reaction with 95% confidence interval. The assay gave a coefficient of variation for the Ct values of <1.4% and 0.78% for intra-assay and interassay, respectively. Taking into account that miR-122 is expressed in >50,000 copies per hepatocyte, this assay is able to suffice the need for reliable detection and quantification of this miRNA. Therefore, this study can be considered as a start point for standardizing miR-122 quantification.
authors: Departments of *Medical Biotechnology †Hematology, Faculty of Medical Sciences, Tarbiat Modares University ‡Nanotechnology and Tissue Engineering Department, Stem Cell Technology Research Center §Department of Immunology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
authors: Mahmood
authors: M
authors: Naderi
authors: Hossein
authors: H
authors: Abdul Tehrani
authors: Masoud
authors: M
authors: Soleimani
authors: Iman
authors: I
authors: Shabani
authors: Seyed Mahmoud
authors: SM
authors: Hashemi
mesh_terms: D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: C531907:MIRN122 microRNA, human; D035683:MicroRNAs