(M1530-09-53) Development of an Extractionless, Multiplexed Assay for the Direct Detection of miRNAs in Liver Tissue

Purpose: MicroRNA (miRNA) detection has become a powerful tool to understand disease pathology, with an increasing interest in the use of miRNAs as molecular biomarkers. Certain miRNAs, including miR-122, are abundantly expressed in the liver and are considered biomarkers for hepatotoxicity. However, miRNA quantification is almost exclusively conducted through miRNA sequencing or singleplex measurements that require amplification. We developed a panel of convenient multiplexed assays that can directly measure miRNAs associated with liver damage across multiple species

Methods: The detection of miRNAs associated with liver toxicity and/or damage was conducted through probe hybridization followed by detection on the 10-spot 96-well N-PLEX® platform. This format allows high-throughput detection of up to 10 distinct miRNAs per well. Two distinct target probes (upstream and downstream) were designed to contain DNA sequences partially complementary to the target miRNA. The upstream probes had a unique 5′ leader sequence that is complementary to spot-specific capture sequences on N-PLEX plates. The downstream probes have a 3′ biotin for detection. Synthetic miRNAs were used as multiplexed calibrators for the assays. Rodent tissue samples or pelleted human hepatocytes (HepaRG cell line) were homogenized in a lysis solution containing RNase inhibitors, and the lysates were collected. To detect miRNAs of interest in samples, a multiplexed mixture of the upstream and downstream probes (for up to 10 targets at a time) were hybridized to their corresponding miRNAs in 20 µL of sample using a thermal cycler. These products were then hybridized to spot-specific capture oligonucleotides on the surface of 10-spot N-PLEX plates and detected with SULFO-TAG™ labeled streptavidin. Plates were analyzed with an MSD® electrochemiluminescence reader. The assays were first qualified using synthetic RNA templates, followed by assessment with human HepaRG cell line samples and rodent tissue lysates.

Results: Multiplexed probe sets were designed against several liver damage biomarkers and screened against synthetic RNA templates. The hybridization temperature of the assays in the N-PLEX plates was optimized to achieve higher stringency and reduce off-target signal generation. The optimized assays were able to specifically identify target miRNAs with an estimated limit of detection of approximately 100 fM or lower. The miRNAs could be directly quantified from the cultured HepaRG cell line, as well as from mouse liver, kidney, lung, heart, and brain lysates without any extraction steps or amplification. In agreement with prior studies suggesting that miR-122 is a liver-specific miRNA, miR-122 levels were high in the liver (820 pM), but not detected in other mouse tissues other than low levels in the kidney (1.7 pM). This is similar to what was observed for miR-192, though concentrations in the liver and kidney were similar to each other (~50 pM). Most other tested miRNAs were present in the liver as well as other tissues, including miR-23a, miR-15a, miR-194, miR-21, etc. Interestingly, in the five tissues tested, miR-125b concentrations in the liver were lower than in the other tissues (1.7 pM), with very high concentrations present in the brain (135 pM). In the HepaRG human hepatocyte cell line, miR-122 and miR-21 had the highest concentrations, whereas others including miR-192, miR-23a, miR-15a, miR-194, miR-193a, and miR-148a had slightly lower concentrations, but they were still readily detectable.

Conclusion: We demonstrated multiplexed measurement of liver damage-associated biomarker miRNAs in human and rodent samples.  The assays do not require RNA extraction and do not use a polymerase-based detection system, allowing simple workflows and a time to result of three to four hours. In a one-plate run, up to 10 miRNAs can be measured from 40 samples if run in duplicate, or 80 samples if run in singlicate, providing a method that is both quantitative for miRNA and amenable to high-throughput screening.