Analytical Validation of Novel microRNA Panel for Risk Stratification of Cognitive Impairment

Abstract

AbstractBackgroundWe have been developing a novel approach to identify risk of early cognitive impairment in patients by profiling of brain‐enriched and inflammation‐associated microRNA (miRNA) biomarkers, isolated from the plasma of cognitively normal and cognitively impaired patients. Here, we present the analytical validation of a miRNA panel, (CogniMIR®), for the expression of 24 miRNAs for large‐scale testing of plasma specimens in a clinical laboratoryMethodmiRNA from plasma specimens was isolated using the MagMAX mirVana kit (ThermoFIsher). 24 synthetic miRNAs oligos (IDT) werecombined and diluted to create quantification markers for making standard curves. RT was performed using miRCURY LNA RT kit. qPCR for miRNA profiling was carried out using custom plates (Qiagen), pre‐plated with lyophilized miRCURY LNA‐based primers for 24 miRNAs of the (CogniMIR®) panel. qPCR was perfomred on a Quantstudio 7 Flex instrument.ResultSynthetic RNA dilution series for 24 miRNAs (200,000 copies, 20000 copies, 2000 copies, 200 copies and 20 copies in a qPCR reaction) were tested on two different days. Average slope of the standard curves for all 24 miRNAs were determined to be in ‐3.3 to ‐3.7 range, and average R2 was 0.99 to 1. The data demonstrates that all 24 synthetic miRNAs of the (CogniMIR®) panel can be reliably and consistently detected even if the sample input is 20 copies in a qPCR reaction.Intra‐run repeatability analysis, using plasma samples from four subjects was tested by two operators on two different days. The data demonstrated that both operators generated repeatable and consistent Cts in both replicates on both days as seen by R2 value of 0.94 to 0.99. The inter‐run data analysis, using the same four samples tested by two operators on two different days, showed that reproducible Ct data were obtained by both operators when the experiment was repeated on the next day as demonstrated by R2 value of 0.96 to 0.97.ConclusionThese results indicate the utility of the (CogniMIR®) panel for miRNA profiling, and demonstrate that our miRNA detection technology is reliable, reproducible, and operationally friendly to test a large scale of specimens in any CAP/CLIA certified clinical laboratory.