Abstract
BackgroundCirculating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is one common approach used to quantitate levels of microRNAs in samples that includes the use of a standard curve of calibrators fit to a regression model. Guidelines are needed for setting assay quantitation thresholds that are appropriate for this method and to biomarker pre-validation.ResultsIn this report, we develop two workflows for determining a lower limit of quantitation (LLOQ) for RT-qPCR assays of microRNAs in exploratory studies. One workflow is based on an error threshold calculated by a logistic model of the calibration curve data. The second workflow is based on a threshold set by the sample blank, which is the no template control for RT-qPCR. The two workflows are used to set lower thresholds of reportable microRNA levels for an example dataset in which miR-208a levels in biofluids are quantitated in a cardiac injury model. LLOQ thresholds set by either workflow are effective in filtering out microRNA values with large uncertainty estimates.ConclusionsTwo workflows for LLOQ determinations are presented in this report that provide methods that are easy to implement in investigational studies of microRNA safety biomarkers and offer choices in levels of conservatism in setting lower limits of acceptable values that facilitate interpretation of results.
Highlights
Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development
A common approach for measurement of miRNA biomarker candidates by absolute quantitation is the use of Reverse transcription quantitative polymerase chain reaction (RT-qPCR) to assay standard curves of synthetic RNA calibrators in parallel to samples for interpolation of unknowns
Calibration curves should cover the dynamic range of the assay, which is a minimum of three orders of magnitude for PCR and ideally five to six [7]. miRNA can be quantified using droplet digital PCR, an alternate PCR-based approach that doesn’t rely on standard curves [8, 9] and is just beginning to be applied to miRNA safety biomarker assessments
Summary
Circulating microRNAs are undergoing exploratory use as safety biomarkers in drug development. Reverse transcription quantitative polymerase chain reaction (RTqPCR) assays have been adapted for the detection of very low levels of miRNAs that are typically found in Wolfinger et al BMC Biotechnology (2018) 18:6 values to a control or reference sample Cq) is not an optimal approach because baseline levels of many candidate tissue-selective miRNAs are very low or undetectable in biofluids and because there is no consensus set of small RNAs in serum or plasma that can be widely used for normalization [4]. A common approach for measurement of miRNA biomarker candidates by absolute quantitation is the use of RT-qPCR to assay standard curves of synthetic RNA calibrators in parallel to samples for interpolation of unknowns. Calibration curves should cover the dynamic range of the assay, which is a minimum of three orders of magnitude for PCR and ideally five to six [7]. miRNA can be quantified using droplet digital PCR, an alternate PCR-based approach that doesn’t rely on standard curves [8, 9] and is just beginning to be applied to miRNA safety biomarker assessments
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