Quantification of miRNAs Co-Immunoprecipitated with Argonaute Proteins Using SYBR Green-Based qRT-PCR

Abstract

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 C _t values obtained from the qRT-PCR assays and enable us to quantify the relative level of Argonaute-bound miRNA.

Keywords: Argonaute; Immunoprecipitation; SYBR Green; miRNA; qRT-PCR.

Figure 1.. Schematic representation of polyT adaptor qRT-PCR method.

Step 1: poly(A) tail is added to miRNA by poly(A) polymerase. Step 2: cDNA is synthesized by reverse transcriptase using oligo-dT adaptor primer. Step 3: cDNA is amplified by SYBR Green-based qPCR with miRNA-specific and universal PCR primers.

Figure 2.

Workflow of SYBR Green-based qPCR of miRNAs using an exogenous miRNA as an internal control.

Figure 3.. Expression and immunoprecipitation of FLAG-Ago2 WT and Y529E.

a. Expression levels of FLAG-Ago2 WT and Y529E were detected by anti-FLAG antibody. Anti-tubulin antibody was used to detect tubulin as an internal control. Non-tagged MBP was used as a negative control. Western blot analysis was triplicated. b. Immunoprecipitated FLAG-Ago2 WT and Y529E were detected by anti-FLAG antibody.

Figure 4.. The amount of miR-19b bound to FLAG-Ago2 WT relative to Y529E

Relative quantification of the bound miR-19b in FLAG-Ago2 WT and Y529E. FLAG-Ago2 Y529E bound less amount of miR-19b than FLAG-Ago2 WT (p < 0.05), which is consistent with the previous report [12]. Experiments were conducted three times.