Validation of a Bead-Based Multiplex Assay for miRNA Quantification in Rat Liver Inflammation

MicroRNAs (miRNAs) are key biomarkers for the diagnosis, prevention, and treatment of various diseases. Most miRNAs interact with one another to influence disease occurrence, making multiplex detection essential for miRNA analysis. In this study, we developed a quantitative analysis method for nine miRNAs using a bead-based flow cytometry system. The method was validated using calibration curves, working range, precision, accuracy, specificity, and carry-over assessments in accordance with bioanalysis guidelines. We analyzed the expression of eight inflammation-related miRNAs, including miR-21, miR-24, miR-29a, miR-34a, miR-122, miR-125a, miR-146a, and miR-155, using cel-miR-39 as an internal control. To demonstrate the applicability of the proposed method, we analyzed nine miRNAs in the livers of male and female rats administered CpG ODN 1826 and LPS and compared the results obtained using the RT-qPCR. The results revealed that the calibration standards for the nine miRNAs included nine points, covering a quantification range of 0.20 to 51.20 pg/μL, with a five-parameter logistic (5-PL) curve fit within ±20%. The quality control (QC) samples for all nine miRNAs met the accuracy and precision criteria, with a %RE within ±20% (25% at the LLOQ and ULOQ) and a %CV of ≤20% (25% at the LLOQ and ULOQ). The total error at each QC level was within the acceptance criteria of ≤30% (40% at the LLOQ and ULOQ). Specificity and carry-over evaluations indicated that specific analytes in the multiplex assay were successfully quantified without interference from other analytes, with no carry-over effects observed. Finally, a total of nine miRNAs were quantitatively analyzed in the livers of rats with inflammation induced by CpG ODN 1826 and LPS, compared with the control group. Of these, the expression of miR-21, miR-34a, miR-146a, and miR-155 tended to increase during liver inflammation in both male and female rats. These results were consistent with the RT-qPCR data. In conclusion, this analytical method, validated in accordance with FDA guidelines, enables accurate and quantitative multiplex detection of inflammation-related miRNA biomarkers in liver tissue. It demonstrates strong reliability in current tissue analyses and provides a foundation for developing multiplex miRNA assays capable of analyzing body fluids, with potential applications in future clinical studies.