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 | •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 | |
•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 | •Avoid false-positive targets outside RISC | •Limited by the specificity of antibody •Low efficiency •Non-specific to miRNA | |
•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 | •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 | |