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Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues

拟南芥叶片组织中线粒体RNA转录水平的分析实验

ED Etienne Delannoy
AL Andéol Falcon de Longevialle
CF Catherine Colas des Francs-Small

发布: 2015年10月20日第5卷第20期 DOI: 10.21769/BioProtoc.1620 浏览次数: 8971

评审: Tie LiuElias BassilAnonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Plant Physiology, featuring study using the protocol.

Aug 2014

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Abstract

This qPCR-based assay provides an overview of the expression levels of all mitochondrial transcripts (mRNAs and rRNAs) as well as splicing efficiency in Arabidopsis. It was developed before RNAseq techniques were widely used (de Longevialle et al., 2007), but is nevertheless still useful as it is cheaper to run and the analysis is much easier and faster to perform if the aim is only to look at mitochondrial transcripts. For intron-containing mRNAs, the use of primer sets specifically amplifying spliced or unspliced forms allows the evaluation of the splicing efficiency.

Keywords: Mitochondrial transcriptome (线粒体转录组) search Splicing assay (拼接法) search Quantitative RT-PCR (定量RT-PCR) search Mitochondria (线粒体) search Arabidopsis thaliana (拟南芥) search

Materials and Reagents

  1. Eppendorf-type microtubes (0.5 ml and 1.5 ml) (any brand)
  2. LightCycler® 480 Multiwell plate (white) (Roche Diagnostics, catalog number: 04729749001 )
  3. LightCycler® 480 Sealing Foil for qPCR run (Roche Diagnostics, catalog number: 04729757001 )
  4. Adhesive PCR film seals for storing plates containing primer mixes (Thermo Fisher Scientific, catalog number: AB0558 )
  5. Combitips 0.1 ml (Eppendorf, catalog number: 0030 089.405 or 0030 089.618 )
  6. Arabidopsis thaliana plants grown either in vitro or in soil
  7. RNeasy Plant Mini Kit (QIAGEN, catalog number: 74904 )
  8. Water, for molecular biology, DNAse, RNAse and Protease free (Thermo Fisher Scientific, ACROS OrganicsTM, catalog number: 327390010 )
  9. Ambion Turbo DNA-freeTM Kit (Life Technologies, catalog number: AM1907 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: AM1907”.
  10. 3M Na acetate (pH 5.2)
  11. 100 % and 70 % ethanol
  12. Agarose LE, analytical grade (Promega Corporation, catalog number: V3125 )
  13. Primers for PCR and qPCR (see Table 1)

    Table 1. Primers used in this protocol
    Please click here for Table 1.

  14. Taq polymerase
    Note: any Taq polymerase can be used for this step, we use a home-purified enzyme.
  15. dNTP set (Life Technologies, catalog number: 10297-018 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10297-018”.
  16. Superscript III Reverse transcriptase (Life Technologies, catalog number: 18080-044 )
    Notes: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 18080-044”.
    This enzyme comes with 5x transcription buffer and a solution of 0.1 M DTT.
  17. Random Hexanucleotide Primers (Life Technologies, catalog number: 48190011 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 48190011”.
  18. RNaseOUT Recombinant Ribonuclease inhibitor (Life Technologies, catalog number:
    10777-019 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10777-019”.
  19. LightCycler® 480 SYBR I Master mix (Roche Diagnostics, catalog number: 04887352001 )
    Note: This reagent is light sensitive.

Equipment

  1. Nanodrop UV-Vis Spectrophotometer (Thermo Fisher Scientific)
  2. ‘Multipette plus’ (Eppendorf, catalog number: 4981 000.019 )
  3. LightCycler® 480 System (Roche Diagnostics)

Software

  1. LightCycler® 480 Software version 1.5 (Roche Diagnostics, catalog number: 04994884001)

Procedure

English
中文翻译

文章信息

版权信息

© 2015 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:

  1. Delannoy, E., Falcon de Longevialle, A. and Francs-Small, C. C. D. (2015). Mitochondrial RNA Transcript Analysis Assay of Arabidopsis Leaf Tissues. Bio-protocol 5(20): e1620. DOI: 10.21769/BioProtoc.1620.
  2. Colas des Francs-Small, C., Falcon de Longevialle, A., Li, Y., Lowe, E., Tanz, S. K., Smith, C., Bevan, M. W. and Small, I. (2014). The pentatricopeptide repeat proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 are involved in the splicing of the multipartite nad5 transcript encoding a subunit of mitochondrial complex I. Plant Physiol 165(4): 1409-1416.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物分子生物学 > RNA

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