The miRNA Target IP Kit was designed to identify the physical interactions of a miRNA with endogenous mRNA transcripts. It utilizes protein G-coupled magnetic beads and a pan-Ago antibody to efficiently immunoprecipitate miRNA/mRNA complexes that have associated with Ago1, Ago2 or Ago3 proteins as part of a RISC complex.
Following their immunoprecipitation, the mRNA targets are purified and then amplified by qRT-PCR using gene-specific primers. This makes it possible to validate the specific mRNA transcripts that are binding targets of that miRNA. It is also possible to profile the immunoprecipitated mRNA molecules using conventional expression microarrays or RNA-Seq.
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miRNA-mediated regulation by RISC complexes
MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules that act as post-transcriptional regulators of gene expression by binding to the 3-prime Untranslated Region (3´UTR) of messenger RNA transcripts (mRNAs). Most commonly, the interaction of a miRNA with a 3´UTR results in either transcript degradation or inhibition of translation.
The targeting of a miRNA molecule to a specific mRNA is mediated through the formation of an RNA Induced Silencing Complex (RISC). Key components in the RISC complex are an Argonaute protein (Ago) and a miRNA. The Ago protein binds the miRNA, positioning it in a conformation that enables the RISC complex to base-pair in a Watson-Crick manner with a mRNA transcript. The sequence of the miRNA molecule contained in a RISC complex enables the precise targeting of specific mRNA transcripts (Figure 1).
Figure 1: Translational repression by a miRNA-directed, RISC Complex.
Validate miRNA/mRNA interactions using Ago IP
The miRNA Target IP Kit utilizes protein G-coupled magnetic beads and a pan-Ago antibody that recognizes Ago 1, Ago 2 and Ago 3 proteins to identify the physical interactions of miRNAs with endogenous mRNA transcripts. The kit supplies sufficient reagents to perform 10 miRNA Target IP reactions using the included Ago1/2/3 Antibody and 10 negative control IP reactions using the included Negative Control IgG. Following their immunoprecipitation as part of the RISC complex, the mRNA targets can be purified and then amplified by qRT-PCR using gene-specific primers. By comparing cells transfected with or without a miRNA mimic, one can identify the targets of a particular miRNA based on over-expression of that miRNA (Figure 2).
Figure 2: The miRNA Target IP Kit shows that miR-122 targets G6PC3 and SLC1A5.
Contents & Storage
Please note that Active Motif's miRNA Target IP Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please store each component at the temperature indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. Do not re-freeze the Protein G Magnetic Beads after you have received this kit. This kit includes the following components:
- Ago1/2/3 antibody (mAb) (1.0 µg/µl); Store at -20°C
- 1 M DTT; Store at -20°C
- RNase Inhibitors; Store at -20°C
- Protease Inhibitor Cocktail (PIC); Store at -20°C
- Ribonucleoside Vanadyl Complex; Store at -20°C
- BSA powder; Store at -20°C
- Proteinase K (10 µg/µl); Store at -20°C
- Precipitation Buffer; Store at -20°C
- Glycogen (20 µg/µl); Store at -20°C
- IGF2 Primer pair (2.5 µM each); Store at -20°C
- Protein G Magnetic Beads; Store at 4°C
- 10X PBS; Store at 4°C
- Negative Control Mouse IgG (0.2 µg/µl); Store at 4°C
- Lysis Buffer AM6; Store at 4°C
- 10X Wash Buffer AM7; Store at 4°C
- 0.5 M EDTA; Store at RT
- 10% SDS; Store at RT
- Salt Solution; Store at RT
- Bar magnet; Store at RT
- Glue dots (2 sheets); Store at RT
Some select publications are listed below.
- “Polyamines release the let-7b-mediated suppression of initiation codon recognition during the protein synthesis of EXT2.” by Imamura et al. (2016) Scientific Reports 6(33549).