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

RNA Stability Measurements Using RT-qPCR in Arabidopsis Seedlings

RT-qPCR法测定拟南芥幼苗RNA稳定性

TJ Tianran Jia
BL Brandon H. Le

发布: 2020年07月20日第10卷第14期 DOI: 10.21769/BioProtoc.3680 浏览次数: 7403

评审: Zhibing LaiAntony ChettoorJason Liang Pin Ng

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 The Plant Cell, featuring study using the protocol.

Oct 2017

Advertisement

Special Issue on Protocols for Organoid Research
Technologies for RNA Detection
“科研可重复性与创新”系列网络研讨会
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

对拟南芥样本的cDNA 3'末端进行快速扩增（3' RACE）

对拟南芥样本的cDNA 3'末端进行快速扩增（3' RACE）

Encarnación Rodríguez-Cazorla [...] Antonio Vera

2015年10月05日 21515 阅读

本塞姆氏烟草中PAMP诱导标记基因表达的实时定量PCR分析

本塞姆氏烟草中PAMP诱导标记基因表达的实时定量PCR分析

Fan Liu [...] Yuanchao Wang

2018年10月05日 9505 阅读

拟南芥子房中轴与侧区的激光辅助显微切割及高通量RNA测序

拟南芥子房中轴与侧区的激光辅助显微切割及高通量RNA测序

Valentín Luna-García and Stefan de Folter

2024年09月05日 2274 阅读

Abstract

Steady-state mRNA levels are determined by both the rates of transcription and degradation. Regulation of mRNA stability and/or degradation are key factors that can significantly affect mRNA levels and its biological functions. mRNA stability can be measured indirectly after transcription inhibition. This protocol described a rapid and sensitive method of mRNA stability measurement through quantitative reverse transcription PCR (RT-qPCR) after inhibition of RNA transcription by cordycepin in Arabidopsis seedlings.

Keywords: RNA Stability (RNA稳定性) search RNA Degradation (RNA降解) search Arabidopsis (拟南芥) search RT-qPCR (RT-qPCR) search Cordycepin (虫草素) search Primary MicroRNA Transcripts (初级MicroRNA转录本) search

Background

The regulation of mRNA stability is a key control point in gene expression regulation. mRNA stability has a profound impact on gene expression, molecular and cellular phenotype, and ultimately, plant development, defense and other biological processes. Multiple methods, e.g., Northern blot analysis, in situ hybridization, can be used to measure mRNA stability after transcription inhibition. In this protocol, we describe a rapid and sensitive method to measure mRNA stability through RT-qPCR after inhibition of transcription by cordycepin. Cordycepin or 3'-deoxyadenosine is an adenosine analogue (see Reference 2). 3'-deoxyadenosine can be incorporated into RNA and inhibits transcription elongation and RNA synthesis due to the absence of a hydroxyl moiety at the 3' position (see Reference 6). We have successfully used this convenient and sensitive method to measure the stability of several low-abundant mRNAs, including primary microRNA transcripts, in Arabidopsis seedlings (Jia et al., 2017). Here we present this protocol with detailed experimental procedures and data analysis methods.

Materials and Reagents

  1. Supplies
    1. Petri dishes, polystyrene, size 150 mm × 15 mm, sterile
    2. 6-well clear multiwell plates, sterile
    3. KimwipesTM
    4. Low retention tips

  2. Materials
    1. Arabidopsis seeds, including control and treatment or mutant groups
      It is strongly recommended to include a mutant with defects in mRNA stability as a positive control, e.g., ect2-1(SALK_002225).

  3. Reagents
    1. Liquid nitrogen
    2. RNaseZapTM (ThermoFisher, catalog number: AM9780 ) or ELIMINase® (DeconLabs, catalog number: 1101 )
    3. Murashige and Skoog Basal Medium (Sigma-Aldrich, catalog number: M5519 )
    4. Agar (Sigma-Aldrich, catalog number: A1296 )
    5. Household bleach (5.25% w/v sodium hypochlorite)
    6. Tween® 20 (Sigma-Aldrich, catalog number: P9416 )
    7. Nuclease-free water
    8. Cordycepin (Sigma-Aldrich, catalog number: C3394 )
    9. RNA extraction kit (RNeasy Plant Mini Kit) (QIAGEN, catalog number: 74904 )
    10. cDNA synthesis kit (iScript cDNA Synthesis Kit) (Bio-Rad, catalog number: 1708890 )
    11. qPCR kit (SYBRTM Green PCR Master Mix) (ThermoFisher, catalog number: 4309155 )
    12. qPCR primers (Design qPCR primers on Primer3Plus (Untergasser et al., 2012), and order qPCR primers from Integrated DNA Technology)
    Note: Design qPCR primers closer to the 3′ end of the mRNAs to be detected. QIAGEN RNeasy Plant Mini Kit, Bio-Rad iScript cDNA Synthesis Kit and ThermoFisher SYBRTM Green PCR Master Mix were tested and worked well, and therefore listed here only as recommended kits. However, other suitable RNA extraction and RT-qPCR kits could also be used for this protocol.

Equipment

  1. Multichannel pipettes
  2. Mortar and pestle
  3. Autoclave
  4. -20 °C freezer
  5. Arabidopsis growth room or chamber
  6. VWR® Standard Orbital Shaker, Model 3500 (VWR, catalog number: 89032-092 )
  7. Eppendorf Benchtop Centrifuge 5418, non-refrigerated, with Rotor FA-45-18-11 and rotor lid, 230 V/50-60 Hz (Eppendorf, catalog number: 5418000017 )
  8. Applied BiosystemsTM QuantStudioTM 5 Real-Time PCR System (ThermoFisher, catalog number: A34322 ), or Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad, catalog number: 1855196 )

Software

  1. Primer3Plus (A. Untergasser, https://primer3plus.com/cgi-bin/dev/primer3plus.cgi)
  2. Excel (Microsoft)
  3. Applied BiosystemsTM Analysis Software or Thermo Fisher Cloud (Thermo Fisher Scientific, available here)

Procedure

English
中文翻译

文章信息

版权信息

© 2020 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Jia, T. and Le, B. H. (2020). RNA Stability Measurements Using RT-qPCR in Arabidopsis Seedlings. Bio-protocol 10(14): e3680. DOI: 10.21769/BioProtoc.3680.

ris ris Download Citation in RIS Format

分类

植物科学 > 植物分子生物学 > RNA > 转录

生物化学 > RNA > RNA结构

您对这篇实验方法有问题吗？

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