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While there is considerable promise in the use of miRNA biomarkers in biofluids, the precise quantification of circulating miRNAs is challenging and the validation of introduced biomarkers is often unsuccessful. This is indicated by a decline in published biofluid-based miRNA biomarker research in the past couple of years, whereas between 2009 and 2015 yearly publications nearly doubled (Chorley et al. 2021a). This downward trajectory is partially attributed to technical obstacles for measuring miRNAs, some of which have been discussed in the preceding sections. Pre-analytical variables such as sample collection, processing, storage, and extraction are potential causes of inconsistency in miRNA measurements. Post-analytical challenges include the normalization of data and the interpretation of miRNA changes observed to be due to perturbation and release into biofluids. These uncertainties and challenges have inhibited full implementation in multiple sectors such as clinical, regulatory, and academic use.

For pre-analytical steps, the standardization of procedures and quality assurance will significantly reduce measurement variability. These considerations extend to consistency in sampling during normal cyclic patterns (circadian, menstrual, etc.), location and fraction of sampling (for example in blood: sublingual, aortic, jugular, etc. and whole blood, serum, plasma, or EV fraction), minimization of hemolysis of collected blood samples, and short-term and long-term storage considerations. Also, miRNAs in biofluids may vary in stability owing to interactions with RNA-binding proteins, EVs, and lipoproteins that may impart protection from high RNase activity in these matrices. In freshly isolated human serum, miRs-16, -21, and -142–3p demonstrated greater stability than miRs-122 and -1 after twenty-four hours at room temperature (Koberle et al. 2013). This difference was affected by RNase activity in the serum, whereas miRNAs in EVs were protected against this activity.

If isolating EVs, the techniques used can influence the content measured. First, there is confusion in the literature on how to distinguish different subclasses of EVs, and this subsequently complicates the identification of RNA cargo (Mathieu et al. 2019). Further, RNA can bind other types of carriers such as lipoprotein and ribonucleoproteins, which are difficult to separate from membrane-enclosed RNAs (O’Brien et al. 2020). Not surprisingly, vesicle purification can lower the RNA yield and integrity and, because of the lower RNA concentration, result in more interindividual variability and increased difficulty in measuring some miRNA biomarkers. To compound the issue, obtaining enough miRNA from vesicles is also a challenge, as studies have demonstrated an average of one or less than one miRNA per EV (Chevillet et al. 2014; Li et al. 2014). Therefore miRNA biomarkers released through active and passive mechanisms may have different stability levels in stored samples and recovery may be inherently low, requiring optimization and consistency in sample collection for specific miRNAs. There are a variety of methods that can be used to measure miRNAs in biofluids, and the selection depends on application, cost considerations, expertise, and available resources. These methods include lower-throughput techniques such as quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or digital drop PCR (ddPCR), medium-throughput techniques such as bead hybridization (e.g., NanoString™ or FirePlex™ assays), and higher-throughput techniques such as microarray and next generation sequencing. A comparative study that used qRT-PCR across eleven research institutions to examine miRNA biomarker alterations due to toxicant exposure found that, when outliers were removed, miRNA measurements were very consistent (only contributing 2% of the measured variance). This supports the idea that standardized protocols can lead to consistent measurements regardless of different site locations (Thompson et al. 2016). Comparison across methods shows consistent correlation when using fold change calculations: concordance ranges from 0.6 to 0.95 (Giraldez et al. 2018; Git et al. 2010; Mestdagh et al. 2014; Yauk et al. 2010).

While miRNAs can be consistently measured methodologically, a primary post-analytical challenge is data normalization. The normalization of measured miRNA data would theoretically correct for natural inter-individual fluctuations in fluid volume and rhythmic or cyclic release, as well as for technical variations attributed to miRNA extraction methods, assay inhibition due to biological factors, and other variables. This would allow precise site-to-site, temporal, and methodological comparisons, alongside increasing confidence in interindividual evaluations. Endogenous “housekeeping” calibrators measured in biofluids such as miRNAs or other nucleic acids would be ideal; however, no universally invariant calibrator has been found (Saliminejad et al. 2019). Where endogenous normalizers may not be available or optimized, synthetic exogenous normalizers or miRNAs “spiked-in” during or after small RNA extraction are commonly used to correct technical variations introduced during sample processing and measurement. A recent study analyzed the intra- and inter-individual variability of circulating miRNAs among healthy and cancer patients (Vigneron et al. 2016). Intra-individual variability significantly improved with a geometric mean of three exogenous measurements by comparison with endogenous normalization. These same normalizers—and, interestingly, the endogenous normalizer miR-16-5p—also improved cross-platform correlation of qRT-PCR and microarray measurements. Much like pre-analytical variables, normalization procedures must be optimized for each individual system and application to increase one’s confidence in the measurements taken.

Recently the microRNA Biomarkers Working Group of the Health and Environmental Sciences Institute’s (HESI) Committee on Emerging Systems Toxicology for the Assessment of Risk (eSTAR) examined the pre- and post-analytical variables that have impeded the development of miRNA biomarkers for toxicological and regulatory use (Chorley et al. 2021a). As a result of examining both the technical and the biological variables that influence the measurement of miRNA biomarkers in biofluids, the group made six recommendations for future research. These recommendations were: determining the pre-analytical stability of miRNAs; establishing standard operating procedures for collection and measurement; establishing a reporting framework; determining the mechanisms of action of extracellular release of miRNAs; determining the linkage of miRNA variants (also called isomeric miRNAs or “isomiRs”) to adverse outcomes; and identifying the cellular or tissue source of extracellular miRNAs. Establishing these unknowns for a miRNA biomarker application strengthens one’s confidence not only in the data produced but also in the mechanism by which the miRNA is released into the biofluid, thereby indicating clear linkages to the adverse outcome of interest and an increased specificity and sensitivity of the biomarker. Of the recommendations, the establishment of standard operating procedures was the highest priority. Procedures that follow standard guidelines for biofluid collection, storage, and handling (such as those outlined by the Early Detection Research Network (ERDN)), as well as the automation of procedures, where applicable, will ensure consistent, reproducible results (Farina et al. 2014).