Establishment of a direct quantitative method for measurement of microRNA-224 in serum by UHPLC/MS/MS

MicroRNAs (miRNAs) are known as potential noninvasive biomarkers for cancer diagnosis. Previous studies have been reported that miR-224 is upregulated in hepatocellular carcinoma (HCC) tissues and sera samples. However, current available methods of miRNA detection typically require pre-enrichment, amplification and labeling steps, and mostly they are semi-quantitative. Herein, we developed an isotope dilution mass spectrometry approach to convert the signal of miR-224 into the mass response of a reporter peptide. Specifically, the newly formed DNA-peptide probe was hybridized with miR-224, which was biotinylated and attached to streptavidin agarose in advance. After through trypsinization, solid phase extraction and blow drying, it used a UHPLC/MS/MS-based quasi-targeted proteomics assay for miR-224 quantification and determines the peak area or peak height ratio of labeled and non-labeled analytes to which an isotope label was added to estimate the concentration of the analyte in the sample. Moreover, this method showed good linearity, precision, accuracy and recovery in the calibration range. In addition, it also demonstrated good performance in comparison with qRT-PCR. Taken together, this study may offer a novel direct quantitative method for serum miRNA analysis applied in clinical practice.