使用miRNA抑制进行miRNA功能和基因调控的研究

  • 可直接用于转染的抑制剂,结合和抑制miRNAs
  • 基于miRBase中人类、小鼠和大鼠的miRNAs设计
  • 可灵活选择规格、包装和纯度
  • 为miRBase中未列出的miRNAs提供订制设计
miScript miRNA Inhibitors是化学合成的、经修饰的单链RNAs,转染到细胞后,可特异性抑制内源性miRNA的功能。提供细胞培养级纯度(>90%纯度)或动物级纯度(经HPLC纯化;用于体内实验)的miRNA抑制剂。另有经硫代磷酸酯修饰的miScript miRNA Inhibitors,可进行体内应用,提高分子的稳定性,规格为20 nmol。如需进行灵活的筛选实验,可选择miScript miRNA Inhibitor Plates,以96孔板或384孔板形式提供预制和用户定制miScript miRNA Inhibitors。在类似实验中,也可使用miScript miRNA Mimics。
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  • 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)

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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
219300 Varies
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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 Inhibitors经修饰后,用于有效抑制内源性miRNA的功能(参见"miScript miRNA Inhibitor counteracts miRNA-induced silencing")。转染miScript miRNA Inhibitor后,Real-time PCR分析可检测到miRNA水平的显著下降(参见Transfection of miRNA inhibitor decreases endogenous miRNA level)。Time-course实验表明,miScript miRNA Inhibitors在转染后,能有效抑制至少96个小时(参见"miRNA inhibitor effective after 96 hours")。
Principle

抑制剂转染及下游基因表达分析或表型分析,用于确定特定miRNAs的靶向和作用。这些实验能够研究个体miRNAs失调的生物效应及确定作为个体miRNAs靶向的特异性基因。

抑制剂转染后基因表达增强表明研究中的miRNA由该基因调控。类似的研究也可使用miScript miRNA Mimics。一般而言,这样的实验都要涉及到模拟物或抑制剂的转染或与携带miRNA结合位点融合报告基因的载体结构共转染。下游分析包括报告分析、real-time PCR、微阵列分析或蛋白质分析。

Procedure

miScript miRNA Inhibitors提供高度灵活的溶液,适合当前版本microRNA数据库(miRBase)中的人类、小鼠、大鼠和病毒miRNA。QIAGEN's GeneGlobe数据库持续更新数据,确保模拟物和抑制剂与最新的miRBase中数据尽量匹配。确保得到高度准确、当前的miRNA序列信息。若miRBase中没有您所需的miRNA的数据,可访问QIAGEN的用户定制miScript产品设计页面,获取适合所需的人类、小鼠、大鼠或病毒miRNA订制合成信息。

可灵活选择规格,用少量抑制剂进行低通量分析或大量抑制剂进行高通量筛查实验。miScript miRNA Inhibitors提供细胞培养级纯度(>90%,由专门的合成和纯化流程提供)的抑制剂。用于体内实验,经HPLC纯化、动物级纯度的miScript miRNA Inhibitors也可订购得到,规格为20 nmol(以试管形式提供)。另有经硫代磷酸酯修饰的miScript miRNA Inhibitors用于体内实验,规格为20 nmol。硫代磷酸酯修饰避免抑制剂失去外切酶活性,提高分子的稳定性。miScript miRNA Inhibitors可在GeneGlobe上订购。对于中到高通量应用,提供96孔板或384孔板形式,满足您的要求。

Applications
  • miRNA研究
  • 基因调控研究
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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