Biotinylated Micro-RNA Pull Down Assay for Identifying miRNA Targets
采用生物素化Micro-RNA pulldown鉴定miRNA靶标
发布: 2017年05月05日第7卷第9期 DOI: 10.21769/BioProtoc.2253 浏览次数: 18972
评审: HongLok LungChiara AmbrogioAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Apr 2016
Abstract
microRNA (miRNA) directly associates with its target transcripts (mRNA). This protocol describes a method for detection of direct interaction between miRNA and mRNA. The result of interaction helps screening the specific target mRNAs for a miRNA.
Keywords: miRNA (miRNA)
Biotin-pulldown (生物素-pulldown)
miRNA-mRNA interaction (miRNA-mRNA相互作用)
Transfection (转染)
Biotin labelling (生物素标记)
Background
MiRNAs are small non coding regulatory RNAs. MiRNAs typically control the gene expression by binding to complementary sequence in their target mRNAs. Many bioinformatics and computational programs are available to predict miRNA targets. But experimental methods identifying direct association of miRNA with target mRNA are very limited. The current protocol can be very helpful in identifying miRNA targets (Phatak et al., 2016).
Materials and Reagents
- 10 cm tissue culture dishes
- 1.5 ml microfuge tube
- PCR tubes
- 15 and 50 ml polypropylene tubes
- Q-PCR plates
- Cells
- Custom synthesis of miRNA sequence with 3’BIOTIN manipulation
- Phosphate buffered saline (PBS)
- Protease inhibitor (100x cocktail) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 78430 )
- RNase inhibitor (Thermo Fisher Scientific, catalog number: EO0381 )
- Streptavidin-Dyna beads (Dyna beads M-280 streptavidin) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11205D )
- Yeast tRNA (stock 10 mg/ml) (Thermo Fisher Scientific, AmbionTM, catalog number: AM7119 )
- TRIzol (Thermo Fisher Scientific, AmbionTM, catalog number: 10296028 )
- Chloroform (Sigma-Aldrich, catalog number: C2432 )
- 2-propanol (Sigma-Aldrich, catalog number: I9516 )
- GlycoBlueTM coprecipitant (Thermo Fisher Scientific, InvitrogenTM, catalog number: AM9515 )
- 200% proof ethanol (Sigma-Aldrich, catalog number: E7023 )
- Nuclease free water (Quality Biological, catalog number: 351-029-721 )
- Reverse transcription system (Promega, catalog number: A3500 )
- Micro RNA RT Kit (Thermo Fisher Scientific, Applied BiosystemTM)
- miRNeasy Mini Kit (QIAGEN, catalog number: 217004 )
- Qiashredder (QIAGEN, catalog number: 79656 )
- RNAiMax (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778150 )
- Potassium chloride (KCl)
- Magnesium chloride (MgCl2)
- Tris-hydrochloride (pH 7.5)
- IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896 )
- Sodium hydroxide (NaOH)
- Sodium chloride (NaCl)
- Lysis buffer (see Recipes)
- Solution A (see Recipes)
- Solution B (see Recipes)
Equipment
- Magnetic separation Stand Magnesphere® technology (Promega, catalog number: Z5342 )
- Mini tube rotator (Fisher Scientific, model: Fisher ScientificTM Mini-Tube rotators , catalog number: 05-450-127)
- Refrigerated microfuge LegendTM micro 21R (Thermo Fisher Scientific, model: SorvallTM LegendTM 21R )
- ABI Step one plus Q-PCR system (Thermo Fisher Scientific, Applied BiosystemTM, model: StepOnePlusTM Real-Time PCR System , catalog number: 4376599)
- Veriti 96 well Thermal Cycler 0.2 ml alloy (Thermo Fisher Scientific, Applied BiosystemTM, model: Veriti® 96-Well Thermal Cycler , catalog number: 4375786)
- -20 °C freezer
- Vortexer
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Phatak, P. and Donahue, J. M. (2017). Biotinylated Micro-RNA Pull Down Assay for Identifying miRNA Targets. Bio-protocol 7(9): e2253. DOI: 10.21769/BioProtoc.2253.
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分类
生物化学 > RNA > miRNA靶标
分子生物学 > RNA > miRNA与mRNA 的相互作用
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