miScript miRNA Inhibitors

miRNA阻害を利用したmiRNA機能および遺伝子制御に関する研究

  • 内在性miRNAに結合し効果的に阻害する即導入可能なInhibitor
  • miRBase に登録されているヒト、マウス、ラットmiRNA に対応
  • 様々なフォーマット、スケール、グレードで提供
  • miRBaseに登録されていないmiRNAでもカスタムデザインで対応可能

miScript miRNA Inhibitor は修飾された合成一本鎖RNA で、細胞へのトランスフェクション後に内在性のmiRNA 機能を特異的に阻害します。miScript miRNA Inhibitors は、細胞培養グレード(純度90%以上)あるいは動物グレード(HPLC精製、in vivo アプリケーション用)でお届けしています。miScript miRNA Inhibitor は、in vivo 使用でより安定なS- 化修飾(Phosphorothioate)も20 nmol スケールで入手できます。miScript miRNA Inhibitor Plates ではデザイン済みあるいはカスタム合成のmiScript miRNA Inhibitorsを、96 ウェルあるいは384 ウェルプレートからお選びいただけます。miScript miRNA Mimicsを同様の実験に使用可能です。

Search in GeneGlobe
You can search for the following terms:
  • Entrez Gene IDs (e.g., 835)
  • RefSeq IDs (e.g., NM_032983, NP_116765)
  • Gene symbols (e.g., CASP2)
  • Cat. no. (e.g., SI00299551, QT01342509)
  • Sanger ID or Accession (e.g., hsa-let-7b, MI0000063)
  • CpG loci identification numbers (CG#) (e.g., CG17753661)

Do not enter species information in the Search box. Use the drop-down list to select your species of interest.
When searching for miRNAs, do not omit the hyphens. Use hyphens or spaces (e.g., search for hsa-let-7b or hsa let 7b. Do not search for hsalet7b).

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miScript miRNA Inhibitor
1 nmol (cell-culture grade), 5 nmol (cell-culture grade), or 20 nmol (animal grade, option of phosphorothioate modification) miScript miRNA Inhibitor, provided in tubes
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miScript miRNA Inhibitors are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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miRNA Inhibitor effective after 96 hours.|miScript miRNA Inhibitor counteracts miRNA-induced silencing.|Transfection of miRNA inhibitor decreases endogenous miRNA level.|
HeLa S3 cells (6 x 104 cells/well) were transfected using HiPerFect Transfection Reagent with miR-15a Inhibitor, miR-16 Inhibitor, miR-21 Inhibitor, miR-25 Inhibitor, or an Inhibitor with a scrambled sequence (negative control). At the time points indicated, luciferase reporter constructs with the appropriate miRNA binding sites were transfected. A reporter construct that is not regulated by any miRNA was used as positive control. Twenty-four hours later, luciferase activity was measured.|HeLa S3 cells express miR-16 at high levels and do not express miR-1 (see Ref. 1). In a cotransfection experiment using HiPerFect Transfection Reagent, HeLa S3 cells (6 x 104 cells/well) were cotransfected with a luciferase reporter construct with an miR-16 binding site in the 3' UTR together with miR-16 Inhibitor. miR-16 Inhibitor was used at varying concentrations in the experiment to evaluate the optimal inhibitor concentration required to see the inhibitory effect. Alternatively, cells were transfected with miR-1 Inhibitor alone as a control. A luciferase construct without an miRNA binding site (WT) was transfected as a positive control. An increase in luciferase expression following transfection of the Inhibitor indicated that the miR-16 Inhibitor prevented endogenous miR-16 from downregulating the reporter gene.

1. Lagos-Quintana, M. et al. (2001) Identification of novel genes coding for small expressed RNAs. Science 294, 853. |HeLa S3 cells (6 x 104 cells/well) were transfected using HiPerFect Transfection Reagent with miScript miR-16 Inhibitor or a negative control. Endogenous miR-16 levels were quantified by real-time PCR using the miScript System. U6B was used as the reference RNA for data normalization and miR-16 levels were expressed as 2-ΔΔCT values relative to the miR-16 levels in untransfected cells. [A] Transfection of miScript miR-16 Inhibitor strongly reduced the amount of detectable endogenous miR-16. [B] The amplification plots indicated a significant decrease in the level of detectable miR-16 as a result of miR-16 Inhibitor transfection. Dissociation curve analysis (inset) reflects the specificity of the real-time PCR detection using the miScript System.|

Performance
miScript miRNA Inhibitorは、内在性のmiRNA機能の効率的な阻害を実現するようにデザイン、かつ修飾されています (図 “miRNA由来のサイレンシング効果を阻害するmiScript miRNA Inhibitor ”)。miScript miRNA Inhibitorのトランスフェクション後にリアルタイムPCR解析によりmiRNA量の顕著な低下が検出できました(図 “miRNA Inhibitorのトランスフェクションによる内在性miRNA量の低下”)。経時変化実験は、トランスフェクション後少なくとも96時間はmiScript miRNA Inhibitor効果があることを示唆しています(図 “トランスフェクション96時間後も効果があるmiRNA”)。
Principle

Inhibitorのトランスフェクションおよびその後の遺伝子発現解析や表現型の解析は特定のmiRNAの標的遺伝子の同定や機能を解明するために有効です。これらの実験により、個別のmiRNAが標的とする遺伝子の確認や個々のmiRNA間違った制御による生物学的影響を調べることができます。

Inhibitorのトランスフェクション後に遺伝子発現が増大すれば、研究対象のmiRNAがその遺伝子の制御に関与している証拠になります。miRNAの作用を促進する miScript miRNA Mimics も類似する研究目的で使用できます。一般に行なう実験では、Mimicあるいは Inhibitorのトランスフェクション、あるいはレポーター遺伝子を融合したmiRNA結合部位を持つベクターコンストラクトとのコトランスフェクションを行います。ダウンストリーム解析としてはレポーターアッセイ、リアルタイムPCR、マイクロアレイ解析、タンパク質解析があります。

Procedure

miScript miRNA Inhibitorは最新の miRBaseに登録されているヒト、マウス、ラットおよびウィルスmiRNA全てに対応して入手できます。最新のmiRBase に対応したMimic およびInhibitor miRNA をデザインするために、QIAGEN の GeneGlobe データベース は常にアップデートされています。従って入手できる最新かつ正確なmiRNA配列情報で常に実験を行うことができます。目的のmiRNA がmiRBase に登録されていない場合には、お客様のヒト、マウス、ラット、ウイルスmiRNA に対応すInhibitor を、QIAGENのmiScript 製品のカスタムデザインページ でカスタムデザインできます。

様々なフォーマットを取り揃えているので、少数のInhibitorを用いたロースループット解析でも数百のInhibitorを用いたハイスループットスクリーニングも可能です。miScript miRNA Inhibitors は、培養細胞に最適なグレード (独自の合成、精製プロセスにより90% 以上の純度) でお届けします。in vivo用にはHPLC精製した動物実験に最適なグレードのmiScript miRNA Inhibitorを20 nmolの収量で注文できます(チューブ)。miScript miRNA Inhibitorは、in vivoアプリケーション用(20 nmol収量)にS-化修飾(Phosphorothiate)したものも提供しています。S-化修飾はエキソヌクレアーゼ活性からInhibitorを保護し、miRNA Inhibitor分子の安定性を増大します。miScript miRNA Inhibitorもウェブサイト GeneGlobe で注文できます。ミドル~ハイスループットなアプリケーション用には、実験の条件に応じて96ウェルプレート、あるいは384ウェルプレートを提供しています。

Applications
miRNA 研究
遺伝子制御研究
Feature
Specifications
Design Predesigned and custom design avaliable
Format Single tubes, 96-well & 384 well plates
Modification Yes, optional with 20 nmol: phosphorothiate modified for in vivo studies
Scale or yield 1 nmol, 5 nmol, 20 nmol
Sequence provided No
Species Human, mouse, rat

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Images
miScript miRNA Inhibitor effective after 96 hours
miRNA Inhibitor effective after 96 hours.
HeLa S3 cells (6 x 104 cells/well) were transfected using HiPerFect Transfection Reagent with miR-15a Inhibitor, miR-16 Inhibitor, miR-21 Inhibitor, miR-25 Inhibitor, or an Inhibitor with a scrambled sequence (negative control). At the time points indicated, luciferase reporter constructs with the appropriate miRNA binding sites were transfected. A reporter construct that is not regulated by any miRNA was used as positive control. Twenty-four hours later, luciferase activity was measured.
miScript miRNA Inhibitor Counteracts miRNA-Induced Silencing
miScript miRNA Inhibitor counteracts miRNA-induced silencing.

HeLa S3 cells express miR-16 at high levels and do not express miR-1 (see Ref. 1). In a cotransfection experiment using HiPerFect Transfection Reagent, HeLa S3 cells (6 x 104 cells/well) were cotransfected with a luciferase reporter construct with an miR-16 binding site in the 3' UTR together with miR-16 Inhibitor. miR-16 Inhibitor was used at varying concentrations in the experiment to evaluate the optimal inhibitor concentration required to see the inhibitory effect. Alternatively, cells were transfected with miR-1 Inhibitor alone as a control. A luciferase construct without an miRNA binding site (WT) was transfected as a positive control. An increase in luciferase expression following transfection of the Inhibitor indicated that the miR-16 Inhibitor prevented endogenous miR-16 from downregulating the reporter gene.

1. Lagos-Quintana, M. et al. (2001) Identification of novel genes coding for small expressed RNAs. Science 294, 853.

Transfection of miRNA Inhibitor Decreases Endogenous miRNA Level
Transfection of miRNA inhibitor decreases endogenous miRNA level.
HeLa S3 cells (6 x 104 cells/well) were transfected using HiPerFect Transfection Reagent with miScript miR-16 Inhibitor or a negative control. Endogenous miR-16 levels were quantified by real-time PCR using the miScript System. U6B was used as the reference RNA for data normalization and miR-16 levels were expressed as 2-ΔΔCT values relative to the miR-16 levels in untransfected cells. [A] Transfection of miScript miR-16 Inhibitor strongly reduced the amount of detectable endogenous miR-16. [B] The amplification plots indicated a significant decrease in the level of detectable miR-16 as a result of miR-16 Inhibitor transfection. Dissociation curve analysis (inset) reflects the specificity of the real-time PCR detection using the miScript System.