Abstract LB-388: New clinical diagnostic approach for miRNA quantification using the Chlorella virus DNA ligase

Abstract

miRNAs have an immense potential to serve as diagnostic, prognostic and prediction biomarkers in the whole field of oncology. Their utilization in liquid biopsy as a non-invasive diagnostic tool is very promising as well. However, to our knowledge, no miRNA-based diagnostics is currently used in clinical laboratories. We believe the reason is simple - the lack of easy, fast, sensitive and reproducible measurement technology, which would have passed the strict clinical validation criteria. On this count we have optimized a very promising miRNA detection technique, utilizing the enzyme Chlorella virus DNA ligase (SplintR® ligase, New England Biolabs), for clinical use. This two-step method involves ligation of two adjacent DNA oligonucleotides hybridized to a miRNA target, followed by TaqMan probe real-time quantitative PCR (qPCR). We managed to reduce the total procedure time from previous 5 hours to 2 hours and 15 minutes maintaining the high sensitivity (1 amol/µl in original clinical sample) and large dynamic range (7 logs). Moreover, we enabled the use of two PCR detection chemistries - more cost-effective SYBR green or more specific TaqMan probe. Based on this optimized method we developed an assay for detection of miR-142-5p, potential marker of colorectal cancer. We measured miR-142-5p profiles in the real samples of whole blood, plasma and serum of healthy donors. The results showed an excellent correlation with the TaqMan qPCR assay and miREIA® immuno-based technology for absolute quantification of miRNA. We conclude, that using SplintR® ligase for miRNA detection is potentially useful for clinical diagnostics. This work was funded by the Ministry of Industry and Trade of Czech Republic, project No. CZ.01.1.02/0.0/0.0/16_084/0008832. Citation Format: Iveta Hynstova, Tereza Mrackova, Michaela Adamcova, Barbora Dvorakova, Zlata Stastna, Ondrej Slaby, Milan Bartos, Viktor Ruzicka. New clinical diagnostic approach for miRNA quantification using the Chlorella virus DNA ligase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-388.