通过干扰miRNA与靶基因间的相互作用,进行miRNA功能研究

  • 为miRNA的发现提供创新工具
  • 在miRNA调控实验中可直接转染
  • 对单个靶基因特异性保护,可准确进行功能研究
  • 提供用户订制服务,保护人类、小鼠或大鼠靶基因

miScript Target Protectors是经修饰的单链RNAs,可通过阻止某个miRNA与它的靶基因结合而实现特异性干扰,同时不会影响这个miRNA对其它靶基因的调控。miScript Target Protectors转染后,可分析表型或基因表达的变化,由此可研究单个靶基因以及特定miRNA的功能。

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miScript Target Protector
5 nmol cell-culture–grade target protector
219000 Varies
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miScript Target Protectors are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Reliable miRNA inhibition.|Target verification.|miScript Target Protectors are specific for the gene of interest.|Inhibition of miRNA regulation of BCL-2.|Phenotype changes after miScript Target Protector transfection.|
miR-16 is endogenously expressed in HeLa S3 and MCF-7 cells. A luciferase reporter construct with a binding site for miR-16 was cotransfected with various amounts of a miScript Target Protector for the same binding site. All amounts resulted in significant increase in luciferase expression caused by inhibition of miRNA downregulation. Neg. control: Luciferase construct cotransfected with Negative Control miScript Target Protector. Unregulated: Luciferase construct without the miR-16 binding site. Attractene Transfection Reagent was used for cotransfections.|MCF-7 cells were cotransfected with miR-29a miScript miRNA Mimic (10 nM) plus Negative Control miScript Target Protector (Mimic) or miR-29a miScript miRNA Mimic plus a miScript Target Protector (1 µM) specific for the putative miR-29a binding site of DNMT3A (Mimic + target protector). Transfections were performed using HiPerFect Transfection Reagent. Forty-eight hours after transfection, expression analysis of DNMT3A was performed by real-time RT-PCR. Untransfected cells were also analyzed. Negative control: Transfection of AllStars Negative Control siRNA and Negative Control miScript Target Protector.|miR-1 downregulates expression of ADAR and HDAC4 genes. HeLa cells were cotransfected with a miScript Target Protector for the miR-1 binding site of ADAR (1 µM, 0.5 µM, 0.1 µM, or 0.05 µM) and miR-1 miScript miRNA Mimic (10 nM). Transfections were performed using HiPerFect Transfection Reagent. After 72 hours, ADAR expression [A] and HDAC4 expression [B] was detected by western blotting. Tubulin was detected as an internal control. Untransfected cells were also analyzed (UT). Negative control (Neg.): Cotransfection of Negative Control miScript Target Protector and miR-1 miScript miRNA Mimic. The miScript Target Protector for the miR-1 binding site of ADAR specifically protected ADAR from miR-1 mediated downregulation and did not affect downregulation of HDAC4 by miR-1.|MCF-7 cells were cotransfected with a miScript Target Protector for the miR-15a/miR-16 binding site of BCL-2 (500 nM or 1 µM) and miR-16 miScript miRNA Mimic (10 nM). After 72 hours, BCL-2 expression was detected by western blotting. Cells transfected with mimic and target protector showed an increase in expression compared to the negative control. Negative control (Neg.): Cotransfection of miR-16 miScript miRNA Mimic and Negative Control miScript Target Protector. Tubulin was also detected as an internal control. Transfections were performed using HiPerFect Transfection Reagent.|MCF-7 cells were transfected with 10 nM miR-9 miScript miRNA Mimic or cotransfected with 10 nM miR-9 miScript miRNA Mimic plus 1 µM miScript Target Protector for the miR-9 binding site of the cell viability gene. After 72 hours, cell viability was examined by light microscopy. Transfections were performed using HiPerFect Transfection Reagent.|
Performance
特异性抑制miRNA对单个靶基因的调控

经修饰的miScript Target Protectors确保特异性、有效结合到目的miRNA结合位点。转染miScript Target Protector后,荧光素酶报告结构实验确保miRNA抑制的可靠性和可重复性(参见"Reliable miRNA inhibition")。miScript Target Protectors保护靶基因的miRNA结合位点,同时不会影响这个miRNA对其它靶基因的调控(参见"miScript Target Protectors are specific for the gene of interest")。

可靠验证miRNA-targeted基因

miScript Target Protectors用于验证基因是否被某个特定的miRNA调控。DNMT3A基因编码DNA甲基转移酶3,在发育和分化中起作用。TargetScan预测到DNMT3A的3' UTR上有miR-29a的单个结合位点。转染miR-29a miScript miRNA Mimic使DNMT3A基因表达下调,如real-time RT-PCR中所示,这表明miR-29a使DNMT3A在转染水平上表达下调。转染经TargetScan预测的miR-29a结合位点的miScript Target Protector,DNMT3A表达上升。这些实验表明DNMT3A被经TargetScan预测miR-29a结合位点负调控(参见"Target verification")。

蛋白质水平的miRNA调控

miRNA可使基因表达在转染水平或蛋白质水平下调。miScript Target Protectors可从两个水平抑制miRNA调控。人类的BCL-2可被2种内源性表达的miRNAs调控:miR-15a和miR-16。miR-15a和miR-16使用BCL-2 3' UTR上相同的结合位点。转染miScript Target Protector,使BCL-2表达在蛋白质水平上升(参见"Inhibition of miRNA regulation of BCL-2")。

miRNA调控对表型的影响

生命进程或信号通路受到影响,引起表型变化,如细胞形态变化或细胞活性变化。观察转染miScript Target Protector后细胞表型的变化,表明miRNA和靶基因在信号通路/生命进程中有关键作用。miR-9使对细胞活性有重要作用的基因表达下调。在MCF-7中,转染miR-9 miScript miRNA Mimic,引起细胞大量死亡。然而,miR-9 miScript miRNA Mimic和为基因mir-9结合位点设计的miScript Target Protector共转染,却没有细胞死亡。这表明miScript Target Protecto可抑制miR-9下调,细胞活性基因可以顺利表达(参见"Phenotype changes after miScript Target Protector transfection")。

Principle

miScript Target Protector是一种新型的工具,可有效识别不同基因调控的miRNAs的作用。还可保护特定靶基因的miRNA结合位点。转染后,miScript Target Protector可结合到miRNA结合位点上,阻止miRNA结合到该位点上,避免其使基因表达下调。

Procedure
转染miScript Target Protector后,可进行表型和基因表达分析。靶基因表达增强、信号模式变化或表型改变都表明miRNA和靶基因在目标信号通路或表型中。转染不同的miScript Target Protectors后,通过特定的表型分析确定各种信号通路中miRNAs的作用。

QIAGEN提供的miRNA功能分析产品还包括miScript miRNA Mimics,一种合成的miRNAs,和miScript miRNA Inhibitors,抑制特定miRNA的所有靶基因的功能。miScript miRNA Mimics和Inhibitors配合miScript Target Protectors使用,可有效识别并发现miRNA的功能。

阳性和阴性对照

使用Positive和Negative Control miScript Target Protectors便于实验设置以及结果分析。Negative Control miScript Target Protector与任何已知的哺乳动物基因都不具有同源性。Positive Control miScript Target Protector保护miR-15a和miR-16调控下内源表达的BCL-2。转染Positive Control miScript Target Protector作为阳性对照,以确定实验设置是否正确。

Applications

miScript Target Protectors配合miScript miRNA Mimics和Inhibitors可用于多种miRNA功能研究,包括:

  • 验证miRNA结合位点
  • 通路或生命进程中的miRNA功能研究
  • 研究靶基因调控
  • 微阵列等分析结果的验证
Feature
Specifications
Design Customer design
Format Single tubes
Scale or yield 5 nmol
Sequence provided No
Species Human, mouse, rat

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