Quantification of miRNAs Co-Immunoprecipitated with Argonaute Proteins Using SYBR Green-Based qRT-PCR
MicroRNAs (miRNAs) are small non-coding RNAs that trigger post-transcriptional gene silencing. These RNAs need to be associated with the Argonaute proteins to be functional. This assembly begins with loading of a miRNA duplex, followed by the ejection of one of the strands (passenger). The remaining strand (guide) together with the Argonaute protein forms a ribonucleoprotein effector complex (the RNA-induced silencing complex, RISC). Mutation on the Argonaute protein, if affecting either step of the RISC assembly, impacts the function of miRNAs. Therefore, any observation of decreased miRNA level of mutants will provide insights into the role of those amino acid residues in the mechanical function of the Argonaute protein. In this chapter, we introduce a method to relatively quantify a specific miRNA co-immunoprecipitated with wild type and mutant Argonaute proteins from HEK293T cells, using Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Spiking a synthetic exogenous miRNA as an internal control with RNA extraction prior to cDNA synthesis will normalize the Ct values obtained from the qRT-PCR assays and enable us to quantify the relative level of Argonaute-bound miRNA.
Key wordsqRT-PCR miRNA Argonaute Immunoprecipitation SYBR Green
We thank G. Singh for providing HEK293T cells and reagents for us. This work was supported by the PRESTO from the Japan Science and Technology (JST) Agency (JPMJPR13L7), The Ohio State University Start-up Fund, and The Ohio State University Center for RNA Biology Seed Grant to K.N.
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