Table 2.

Current methodologies to measure miRNA

Method	Strengths	Limitations	Applications	Metric	Dynamic range	Sensitivitya
SYBR-based qPCR	Sensitivity Broad target compatibility Detection reproducibility Common standard operating procedure Cheaper than probe-based technologies	Multi-step workflows Commonly requires purified RNA Input required for many miRNA screenings can be too high for scarce samples Non-specific amplification difficult to measure IsomiR sequences difficult to discriminate	Discovery development, Validation, Safety assessment/Preclinical studies, Clinical	Cycle threshold (Ct)	7 logs	++++
Hydrolysis probe-based (“TaqMan”) qPCR	Sensitivity Broad target compatibility Detection reproducibility Specificity vs. SYBR qPCR Common standard operating procedure	Multi-step workflows Commonly requires purified RNA Input required for many miRNA screenings can be too high for scarce samples Non-specific amplification difficult to measure IsomiR sequences difficult to discriminate	Discovery and development, Validation, Safety assessment/Preclinical studies, Clinical	Cycle threshold (Ct)	7 logs	++++
Digital PCR	Digital quantification without standard curve Sensitivity due to end-point amplification	Multi-step workflows Commonly requires purified RNA Non-specific amplification difficult to measure IsomiR sequences difficult to discriminate	Discovery and development, Validation, Safety assessment/Preclinical studies, Clinical	Copy number	5 logs	++++
Bead hybridization	Can be compatible with biofluids and tissues without RNA purification Low input with amplification	Number of miRNA detected Sensitivity without amplification is poor IsomiR sequences difficult to discriminate	Discovery, Validation, Safety assessment/Preclinical studies, Clinical	Abundance (with curve)	5 logs	++++
Next generation sequencing	Breadth of small RNA species detected Single nucleotide specificity Can distinguish isomiRs	Laborious multi-step workflow Input for workflows too high for scarce samples	Discovery and development	Reads per unit	5–6 logs	++
Digital counting	Lack of amplification bias Digital quantification without standard curve	Input requirements higher than qPCR Generally lower specificity within miRNA families IsomiR sequences difficult to discriminate	Discovery and development, Validation, Safety assessment/Preclinical studies, Clinical	Counts	5–6 logs	++
Microarray	Breadth of miRNA detected	Workflows are labor intensive Input for workflows is high, requires purified RNA Generally lower specificity within miRNA families IsomiR sequences difficult to discriminate	Discovery and development	Relative fluorescence	3–4 logs	+

Abbreviations: miRNA, microRNA; qPCR, quantitative PCR.

Sources: Jensen et al. (2011), Sah et al. (2010), Tan et al. (2015), and Godoy et al. (2019).

^a++++ = <100 copies, +++ = 100–1000 copies, ++ = 1000–5000 copies, and + = ≥5001 copies.