Extraction of Small RNA and qPCR Validation of miRNAs in Vigna mungo
黑吉豆中小RNA的提取和miRNA的定量PCR
发布: 2015年03月05日第5卷第5期 DOI: 10.21769/BioProtoc.1417 浏览次数: 10969
评审: Samik BhattacharyaAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jan 2014
Abstract
Small RNAs like microRNAs (miRNAs), small interfering RNAs (siRNAs) and other noncoding RNAs including snRNA and snoRNA have tremendous impact on eukaryotic gene regulation. Extraction of high quality small RNAs is an important prerequisite for experimental analyses of miRNAs. This will prevent RNA degradation and remove associated contaminations including polyphenols, polysaccharides and other secondary metabolites. In this protocol we describe a simple way to isolate small RNAs from the leaf tissues of Vigna mungo combining the protocols of two commercially available kits with some modifications.
Keywords: MicroRNA (microRNA)
MYMIV (mymiv)
Next Gen Sequencing (下一代基因测序)
Vigna mungo (豇豆蒙哥)
Materials and Reagents
- Vigna mungo MYMIV-resistant recombinant inbred line, VMR84
- mirPremier microRNA isolation kit (Sigma-Aldrich, catalog number: SNC10 )
- Mir-X miRNA FirstStrand synthesis and SYBR qRT-PCR kit (Takara Bio Company, Clontech, catalog number: 638314 )
- 2-mercaptoethanol (Sisco Research Laboratories, catalog number: 1324196 )
- Ethanol (Merck, catalog number: K41540783 )
- Liquid nitrogen and dry ice
- RNase free water (Bangalore Genei, catalog number: 612151181001730 )
- Agarose (Sisco Research Laboratories, catalog number: 0140229 )
- Ethidium bromide (Sisco Research Laboratories, catalog number: 054817 )
- 1x TAE buffer (see Recipes)
- Ethidium bromide stock (see Recipes)
Equipment
- Thermocycler (DNA Engine Cycler, model: PTC-200 )
- Real-time qPCR (Biorad iQ5 Real-Time PCR Detection System)
- Centrifuge (Thermo Fisher Scientific, model: MicroCL21 )
- Electrophoresis apparatus (Bangalore Genei)
- Heat block or water bath
- Mortar and pestle
- NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Paul, S., Kundu, A. and Pal, A. (2015). Extraction of Small RNA and qPCR Validation of miRNAs in Vigna mungo. Bio-protocol 5(5): e1417. DOI: 10.21769/BioProtoc.1417.
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分类
植物科学 > 植物分子生物学 > RNA > RNA 干扰
植物科学 > 植物分子生物学 > RNA > qRT-PCR
分子生物学 > RNA > RNA 干扰
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