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Table 1 Summarization and comparison of current experimental strategies for intracellular target identification of miRNAs

From: Current experimental strategies for intracellular target identification of microRNA

Experimental strategies and references Advantages Limitations
Gene expression profiling through overexpression or inhibition of specific miRNA Luciferase reporter screening systems [25, 26] •A straightforward method to identify direct targets for miRNAs
•High sensitivity
•Easy to adopt
•High costs
•Lack of 3’-UTR libraries
•Low throughput
•Unable to identify non-canonical targets
Microarray analysis [27, 28] •Simultaneous identification of a subset of genes •Difficult to distinguish direct and indirect miRNA targets
•No information about miRNA-mRNA interaction
•High false-positive and false-negative results
Stable isotope labeling by amino acids in cell culture (SILAC) [29, 30] •Easy quantification of protein production through metabolic labeling
Ribosome profiling [31] •Measuring mRNA translation
Immunoprecipitation of RISC with specific antibody Immunoprecipitation (IP) [32, 35] •Avoid false-positive targets outside RISC •Limited by the specificity of antibody
•Low efficiency
•Non-specific to miRNA
Crosslinking and immunoprecipitation (CLIP) [36, 37] •Increase in capture efficiency due to photo-crosslinking
Crosslinking, immunoprecipitation and sequencing of hybrids (CLASH) [19, 40] •Clear information about miRNA-mRNA interaction due to ligation of miRNA-mRNA in RISC •Limited by the specificity of antibody and crosslinking efficiency
•Low efficiency
Pull-down with labeled miRNA mimics 3’-biotinylated miRNA probes [41,42,43] •High efficiency
•Specific to miRNA
•Side effect of 3’-biotinylation on miRNA function
MiRNA crosslinking and immunoprecipitation (miR-CLIP) [22] •Avoid side effect of biotinylation on miRNA function
•Specific to miRNA
•Limited by the specificity of antibody and crosslinking efficiency
•Low efficiency
•Not universal for other miRNAs
Photo-clickable miRNA [47] •A universal strategy for different miRNAs
•Specific to miRNA
•Possible dissociation between photo-clickable miRNA and target mRNAs during pull-down
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