Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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: 9654

评审: Tie LiuElias BassilAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

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

Advertisement

Special Issue on Protocols for Organoid Research
Technologies for RNA Detection
High-throughput T-cell antigen discovery technology platform and its applications
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

Turbo-RIP：基于 TurboID 的 RNA 免疫纯化方法，用于绘制植物生物分子凝聚体中的 RNA 图谱

Turbo-RIP：基于 TurboID 的 RNA 免疫纯化方法，用于绘制植物生物分子凝聚体中的 RNA 图谱

Zhi Zhang [...] Panagiotis Nikolaou Moschou

2026年02月05日 1122 阅读

DiRT v2.0：用于检测植物双顺反子 tRNA-mRNA 转录本的优化流程

DiRT v2.0：用于检测植物双顺反子 tRNA-mRNA 转录本的优化流程

Fei Zheng [...] Rakesh David

2026年06月20日 360 阅读

硬质植物组织（根和地上部）成像型空间转录组学的样品制备

硬质植物组织（根和地上部）成像型空间转录组学的样品制备

Hanhong Liu [...] Mingyuan Zhu

2026年08月05日 374 阅读

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

您对这篇实验方案有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件