Detection of Argonaute (Ago) protein associated MiRNA by combining anti-ago antibody recognition with real-time PCR

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Brian D. Coley (Creator)
The University of North Carolina at Greensboro (UNCG )
Web Site:
Norman Chiu

Abstract: MicroRNAs (miRNAs) are small molecules of noncoding RNA that range between ~19-22 nucleotides in length. In recent years, scientists have observed that these small RNA molecules exist in the extracellular environment within eukaryotic organisms. Furthermore, these microRNA molecules are known to associate with a family of proteins named Argonaute proteins. These microRNA/Argonaute protein complexes are the core of a larger assembly of proteins that compose the RNA Induced Silencing Complex (RISC). The RISC has exhibited the ability to inhibit the translation of, or cleave, its target messenger RNA (mRNA), the latter of which being exclusive only to Argonaute 2 protein (Ago2). It has also been observed in the literature that these Argonaute/miRNA complexes often target genomic regions associated with various cancers in humans and, currently, more than 2000 miRNAs have been discovered and published in the literature. Current methods for analyzing miRNA expression involve total RNA extraction using methods such as ethanol precipitation. However, total RNA extraction does not take into consideration that the major functional component of post-transcriptional inhibition is indeed that Argonaute protein/miRNA complex and not the miRNA alone. In this study, we investigate a novel method to capture and detect the active Ago2/miRNA (miRNP) complex and quantitate associated miRNAs by utilizing an antibody against Ago2 and subsequent application of real-time PCR to successfully capture the active miRNP complex and quantitate the associated miRNAs.

Additional Information

Language: English
Date: 2014
Argonaute 2, MicroRNAs
Non-coding RNA
Gene silencing

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