Table 3.

Advantages and challenges in using miRNAs as diagnostic marker

Advantages	Challenges
MicroRNAs present in various source materials (i.e., whole blood, plasma, serum, blood cells and tissues) and that can be easily isolated and quantified	Liquid biopsies present special difficulties compared with tissue sampling, as miRNA levels are very sensitive to pre-processing and post-processing factors
miRNAs are highly stable in the circulation, minimal or no differences have been found between fresh and frozen specimens, even after repeated freeze-thaw cycles	Specific standard operating procedures (SOPs) for blood collection and plasma/serum preparation are not followed
	No standard endogenous control for the normalization of miRNA levels in blood has been established Tissue-specific housekeeping miRNAs are not suitable and global normalization approach could not be appropriate for normalization of miRNA profiling data because it assumes that the same total amount of miRNAs is expected in all samples and that only a small percentage of miRNAs is differentially expressed, as both up- and downregulated
Several methods have been developed to quantify circulating miRNAs: qRT-PCR, droplet digital PCR, quantitative stem-loop RT-PCR) and chip-based digital PCR, as well as RNAseq and microarrays
Digital PCR (dPCR) provides a quasi-absolute readout or copy number for miRNAs and eliminates the need for standard curves as well as the influence of normalization strategies (at least for assays that have been thoroughly validated)
The availability of powerful approaches for global miRNA characterization and simple, universally applicable assays for quantitation (e.g., qRT-PCR) suggests that the discovery–validation pipeline for miRNA biomarkers will be more efficient than traditional proteomic biomarker discovery– validation pipelines, which typically encounter bottlenecks at the point of antibody generation and quantitative assay development for validation of biomarker candidates	Expression pattern of miRNAs between different liquid biopsies (e.g., platelet-rich plasma, platelet-poor plasma, serum and whole blood) vary due to difference in their method of separation or composition of blood cells in them
	Potential differences in sample/patient number, sampling time, methods for miRNA isolation, quantification, miRNA normalization parameters and co-morbidities could attribute miRNA expression and profiling
Significant number of miRNAs were identified with the potential for becoming targets in order to understand more about disease pathology	Most miRNAs are expressed widely in a non-cell/tissue-specific manner, and they do not differ drastically in level between cases and controls
	Many miRNAs proposed as biomarkers for one disease have been found in association with a bewildering variety of other conditions
	Compared to DNA or RNA-based tests that indicate the presence of a mutation (s), miRNA tests produce results that are difficult to interpret
	At present, the effect of controllable (gender, age, drug assumption etc.,) and uncontrollable (individual genetics, diet, life style etc.,) pre-analytical factor on the miRNA profiling is not established completely
	Very low percentage of hemolysis can elicit a considerable increase in erythrocyte-specific miRNA levels. Therefore, miRNA-based methods could turn out to be not sufficiently accurate to discriminate hemolyzed samples from samples presenting altered erythrocyte-contained miRNA expression due to other conditions
	No consistent results have been obtained regarding differences in column-based miRNA isolation methods, suggesting that a great effort is still needed in comparing different extraction methods and working toward standardization