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miRNA Target IP Kit

miRNAに特異的な標的mRNAを検証

miRNA Target IP Kitは,内在性mRNA転写物とmiRNAの物理的相互作用を特定するためのキットです。本キットは,プロテインG磁気ビーズとpan-Ago抗体を用いて,RNA Induced Silencing Complex (RISC)の一部であるAgo1, Ago2 またはAgo3タンパク質と関連のあるmiRNA/mRNA複合体を免疫沈降します。その結果,miRNAと内在性mRNA転写産物の実際の物理的相互作用を確認することを可能にします。

本キットはマウス由来3T3細胞およびヒト由来293MCF-7HT1080およびHeLa細胞を用いて検証されています。キットには,Ago1/2/3抗体およびnegative Control IgGが同梱されており,miRNA免疫沈降を10回行うのに十分な試薬が含まれています。免疫沈降後,mRNAターゲットを精製し,遺伝子特異的なプライマーを用いてqRT-PCRにより増幅します。これによりmiRNAの結合標的である特異的なmRNA転写物を検証することができます。

 
Name Format Cat No. 価格 (税抜)  
miRNA Target IP Kit 10 rxns 25500 ¥171,000 Buy Now

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).

Diagram showing translational repression by a miRNA-directed, RISC Complex.
Figure 1: Translational repression by a miRNA-directed, RISC Complex.

The key components in a 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 to base-pair in a Watson-Crick manner with a mRNA transcript. This leads to either inhibition of translation (shown) or increased degradation of the targeted transcript.

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).

A graph of data generated with the miRNA Target IP Kit showing that G6PC3 and SLC1A5 are bound by miR-122 while GAPDH
Figure 2: The miRNA Target IP Kit shows that miR-122 targets G6PC3 and SLC1A5.

The miRNA Target IP Kit was used on samples of HT1080 cells that had been transfected with a miR-122 mimic or a non-targeting miRNA control for 8 hours. Following IP using the Ago1/2/3 antibody or Negative Control IgG included in the kit, qRT-PCR was performed on the samples using primers for G6PC3 and SLC1A5, which are known targets of miR-122, and for GAPDH, a common housekeeping gene that is not known to be targeted by miR-122.

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