MicroRNA Northern blotting, precursor cloning, and Ago2-improved RNA interference

Aberrant expression of microRNAs (miRNAs) and processing defects in their biogenesis pathway are a widespread phenomenon in tumors, conveying great importance to the analysis of miRNA expression, regulation, and biogenesis to gain knowledge about their role in cancer. Besides Drosha and Dicer, Argonaute proteins are key players in miRNA processing. In addition to their role as components of the RNA-induced silencing complex (RISC) executing target silencing, Argonautes mediate posttranscriptional regulation of miRNA maturation by creating an additional intermediate processing step, the Ago2-cleaved precursor miRNA (ac-pre-miRNA), and enhancing the production or stability of mature miRNAs. Here, we describe the detection of miRNA levels by Northern blotting and the identification of the 3' end of miRNAs by precursor cloning to accentuate two of the many roles of Argonaute proteins. In addition, we describe a method to optimize RNAi experiments by increasing the efficacy and specificity of target silencing via Ago2 cotransfection.