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Several in vitro methods have proven useful to validate miRNA function, including RNA interference and protein analysis using SILAC.
Over-expression and knockdown functional studies - Over-expression and knockdown of the expression of a specific miRNA in a cell has proven useful to elucidate miRNA function. There are three basic approaches to induce RNA interference: synthetic RNAi and siRNA duplexes, vectors carrying an RNAi cassette expressing shRNA or artificial miRNAs, and in vitro transcription and dicing of dsRNA to generate pools of siRNA.
Related RNA interference topics:
SILAC studies - Although miRNA profiling can show changes based on miRNA activity, it is not always indicative of translation inhibition. Protein analysis examining by mass spectrometry combined with stable isotopic labeling by amino acids in cell culture (SILAC) has shown a strong correlation with miRNA activity. This method relies upon the metabolic incorporation of 'light' or 'heavy' form of the amino acid into the proteins to distinguish differences between cell populations - such as normal and malignant breast tissue.
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