Quantification of microRNA in plasma using probe based TaqMan assays: is microRNA purification required?

Abstract

Abstract Objective Circulating microRNAs are promising diagnostics and prognostics biomarkers in a wide variety of diseases. However, there is a critical reproducibility challenge, which in part may be due to preanalytical factors. MicroRNA purification has been identified as the major contributor to the total intra assay variation, thus we found great interest in recent papers describing methods for direct quantification of circulating microRNAs without the purification step. With one exception, all the studies we identified where a direct quantification of circulating microRNAs had been performed were using SYBR Green chemistry. In our laboratory we use platelet-poor plasma and TaqMan assays for microRNA analysis, and thus we investigated whether we could adapt the procedures for the direct reverse transcription described by these studies to be used with our TaqMan assays. Results We did not achieve valid results by direct quantification of selected microRNAs (miR-92a, miR-16 and miR-126) in platelet-poor plasma using TaqMan assays.

Keywords

• MicroRNA
• Direct plasma RT-qPCR
• Sample preparation
• TaqMan assays