Table 2.

Methods for miRNA validation.

Method	Throughput	Pros	Cons
Northern blot analysis	Low	Length of transcripts observed, possibility of “double-band”	Work-intensive, lack of sensitivity
PCR-based methods	Low	Specific to transcript 3′end, sensitive	Costly for large-scale validation
Ectopic RNA hairpin expression	Low	miRNA biogenesis is directly tested	Work-intensive, impractical for large-scale validation
Association with Argonaute proteins	Low/high	Directly shows interaction with effector proteins	Method is not always specific for miRNAs
Inhibition of miRNA biogenesis pathways	Low/high	Directly shows dependence on biogenesis proteins	Knock-downs are transient and sometimes weak, generating knock-outs is time-consuming
Experimentally identified target sites	Low/high	Directly demonstrates target interaction or repression	Reporter assays are work-intensive
Conservation and population selection pressure	Sequence analysis	No wet-lab experiments required	Non-conserved miRNAs can be functional