Table 3 miRNA quantification technology

qPCR

• Current gold standard for sensitivity and specificity

• No genome-wide coverage

[74,75,76]

Microarray

• Commercially available reagents

• Genome-wide coverage

• Specific probes

• Specialized equipment

• Lack of reproducibility between platforms

• Difficult data normalization

[81,82,83,84,85,86,87]

NGS

• Genome-wide coverage

• Multiple samples may be run in parallel

• Promotes novel miRNA discovery

• Can detect polymorphisms

• Complicated, non-standardized data analysis

[93,94,95,96,97]

Isothermal amplification

• No need for thermocycling equipment

• Can improve existing qPCR, microarray, and NGS methods

• Disadvantages are technique-specific (see below)

[101]

 • Exponential amplification

• High sensitivity

• May require a nicking enzyme, which complicates primer design

[102, 103]

 • Rolling circle amplification

• 1 primer

• Can be optimized for linear or exponential amplification

• Requires 2 enzymes (polymerase and ligase)

• Initial denaturation not performed at room temperature

[105,106,107,108,109]

 • Duplex-specific nuclease signal amplification

• High specificity

• Enzyme is not readily available

[110,111,112,113]

 • Hybridization chain reaction

• No polymerase

• Linear amplification only

[114,115,116,117,118,119,120]

Near-infrared technology

• No autofluoresence

• Minimal photobleaching

• No tedious treatment of sample before or after the test

• Lanthanide probes are not yet commercially available and must be optimized

[132, 140,141,142,143]