同位素示踪法进行酵母RNA的代谢标记

Yuehua Wei

doi:10.21769/BioProtoc.623

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Peer-reviewed

Metabolic Labeling of Yeast RNA with Radioactive Uracil

同位素示踪法进行酵母RNA的代谢标记

Yuehua Wei email

发布: 2013年04月05日第3卷第7期 DOI: 10.21769/BioProtoc.623 浏览次数: 10133

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参见作者原研究论文

The authors used this protocol in:

Cover of The EMBO Journal, featuring study using the protocol.

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

English

中文翻译

文章信息

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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.