A Potential of microRNAs for High-Content Screening

Table 1

Ways to analyse function of miRNA.

Aim	Method	Features
		Pros	Cons
Knock-out of miRNA gene	Homologous recombination/Gene editing with zinc finger nucleases	(i) Precise intervention	(i) Laborious and time consuming
		(ii) Complete loss-of-function	(ii) Simultaneous knock-out of protein encoded by the same transcriptional unit
Knock-down of miRNA	Antisense oligonucleotide; Ribozymes/DNAzymes	(i) Can attack mature miRNA and miRNA precursors	(i) OTEs and unspecific secondary effects may occur
			(ii) Efficiency of knock-down is difficult to predict
		(ii) Stable expression from vector-constructs
			(iii) Multiple knock-downs might be needed to produce a phenotype
		(iii) Commercially available
		(iv) Transfection/transduction of multiple oligonucleotides is possible
	miRNA sponges	(i) Easy design	(i) Efficiency of knock-down is difficult to predict
		(ii) Simultaneous knock-down of multiple miRNAs
			(ii) Incomplete knock-down of individual miRNAs
		(iii) Expression can be verified by fluorescent reporter
Over-expression of miRNA	miRNA mimics	(i) Efficient silencing of target mRNA	(i) Oversaturation of RNAi machinery can lead to secondary effects
		(ii) Oligonucleotides mimicking mature miRNAs or miRNA precursors could be used
		(iii) Transfection/transduction of multiple mimics is possible
		(iv) Stable transfection from vector-constructs
	Conditional release of miRNA from riboswitch constructs	(i) Controlled expression of mature miRNA	(i) Laborious design
		(ii) Possibly less toxic side effects	(ii) Lack of availability
			(iii) Not yet adaptable to high-throughput analysis
Release of target mRNA from repression by miRNA	Target protectors	(i) Release of specific mRNA from regulation by miRNA possible	(i) Target mRNA has to be known