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Isolation to Functional Analyses

ibed process (Figure 1). A primary transcript (pri-mRNA) that can be more than 1,000 nt in length is first produced in the nucleus. An RNA hairpin (precursor miRNA, pre-miR) comprising ~80 nt that includes the mature miRNA results from digestion of pri-miRNA by the double-strand-specific ribonuclease, Drosha [6]. The resulting pre-miRNA is transported to the cytoplasm via a process that involves Exportin-5 [7,8]. The pre-miRNA is further digested by Dicer [6,8] to generate a short, partially double-stranded RNA wherein one strand is the mature miRNA. The mature miRNA is taken up by a protein complex that is similar to, if not identical to, the RNA-Induced Silencing Complex (RISC) that supports RNA interference (RNAi) [9,10] and the bound complex funtions to regulate translation. For more information on miRNA processing and function, see Reagents for Up and Down- Regulation of miRNA Activity in Mammalian Cells, and MicroRNA Profiling by Array Analysis Reveals Critical BioMarkers.

Figure 1. miRNA and siRNA Processing Pathway.


Obtaining Samples Containing miRNAs, siRNAs, and Other Small RNAs

Because of their small size, isolation and detection of miRNA, siRNA, and other small RNA molecules is not trivial. For instance, methods featuring glass fiber filter (GFF) based binding are used routinely for total RNA sample preparation, but most of these methods do not result in quantitativ
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