Metabolic Labeling of Yeast RNA with Radioactive Uracil
同位素示踪法进行酵母RNA的代谢标记
发布: 2013年04月05日第3卷第7期 DOI: 10.21769/BioProtoc.623 浏览次数: 10797
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参见作者原研究论文
The authors used this protocol in:
Aug 2009
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Abstract
To examine gene expression, Northern blot or Real-Time PCR can be used to detect low abundant RNA such as mRNA. However, for high abundant RNAs such as rRNA and tRNA, Northern blot will not be able to discriminate the newly synthesized RNA from total RNA. Therefore, metabolic labeling is necessary to evaluate the expression of rRNA and tRNA genes. In this protocol, I describe a step-by-step method for labeling yeast RNA with radioactive uracil and examine the synthesis of these high abundant RNAs.
Keywords: Autoradiography (放射自显影)
Metabolic labeling (代谢标记)
Tritium (氚)
RRNA (rRNA)
TRNA (tRNA)
Materials and Reagents
- Yeast strain of interest
- RapidGel (500 ml) (Affymetrix, catalog number: 75848 )
- Urea (CO(NH2)2) (Sigma-Aldrich, catalog number: U6504 )
- Dextrose (C6H12O6) (Sigma-Aldrich, catalog number: G7021 )
- Yeast nitrogen base without amino acids (Sigma-Aldrich, catalog number: Y0626 )
- Synthetic dropout supplement without uracil (Sigma-Aldrich, catalog number: Y1501 )
- Uracil (C4H4N2O2) (Sigma-Aldrich, catalog number: U1128 )
- Rapamycin
- TEMED/Tetramethylethylenediamine (C6H16N2) (Thermo Fisher Scientific, catalog number: 110-18-9 )
- Ammonium persulfate (APS) ((NH4)2S2O8) (Sigma-Aldrich, catalog number: A3678 )
- Formamide (CH3NO) (Thermo Fisher Scientific, catalog number: 75-12-7 )
- DEPC/ Diethylpyrocarbonate (O(COOC2H5)2) (Sigma-Aldrich, catalog number: D5758 )
- Bromophenol Blue (C19H10Br4O5S) (Sigma-Aldrich, catalog number: B0126 )
- Xylene Cyanol FF (C25H27N2NaO6S2) (Sigma-Aldrich, catalog number: X4126 )
- [5, 6-3H]-Uracil (PerkinElmer, catalog number: NET368250UC )
- RNA marker (Promega Corporation, catalog number: G3191 )
- Tris base (Thermo Fisher Scientific, catalog number: 77-86-1 )
- Boric acid (Thermo Fisher Scientific, catalog number: 10043-35-3 )
- EDTA (Sigma-Aldrich, catalog number: EDS-1KG )
- Formamide loading dye
- Small RNA separating gel
- SD-Ura- (see Recipes)
- SD-1/3 Uracil (see Recipes)
- DEPC water (see Recipes)
- 10x TBE in DEPC water (see Recipes)
- Small RNA separating gel (see Recipes)
- 2x Formamide loading dye (see Recipes)
Equipment
- Bench top centrifuge
- 30 °C Shaker
- Power supply with constant voltage > 450 V
- Gel dryer (Bio-Rad Laboratories, catalog number: 165-1745 )
- Exposure cosset/intensifier screen (Sigma-Aldrich, catalog number: C5479-1EA )
- 50 ml conical tubes
- 1.5 ml Eppendorf tube
Procedure
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文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Wei, Y. (2013). Metabolic Labeling of Yeast RNA with Radioactive Uracil. Bio-protocol 3(7): e623. DOI: 10.21769/BioProtoc.623.
- Wei, Y., Tsang, C. K. and Zheng, X. F. (2009a). Mechanisms of regulation of RNA polymerase III-dependent transcription by TORC1. EMBO J 28 (15): 2220-2230.
Download Citation in RIS Format
分类
分子生物学 > RNA > RNA 标记
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