A Home-brew Real-time PCR Assay for Reliable Detection and Quantification of Mature miR-122. Issue 8 (September 2015)

• Record Type: Journal Article
• Authors: Naderi, Mahmood; Abdul Tehrani, Hossein; Soleimani, Masoud; Shabani, Iman; Hashemi, Seyed Mahmoud
• Is Part Of: Applied immunohistochemistry & molecular morphology. Volume 23:Issue 8(2015)
• Publication Date: 2015-09
• Subjects: Diagnostic immunohistochemistry -- Periodicals; Immunohistochemistry -- Periodicals; Cells -- Morphology -- Periodicals; Molecular diagnosis -- Periodicals; 616.079
• DOI: 10.1097/PAI.0000000000000125
• Languages: English
• ISSNs: 1541-2016

Abstract

<abstract> <title> <x xml:space="preserve">Abstract</x> </title> <sec> <p>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<sup>6</sup> to 4.8E<sup>3</sup> 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 <italic>C</italic><sub>t</sub> 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.</p> </sec> </abstract>