RT-qPCR法测定拟南芥幼苗RNA稳定性

Tianran Jia; Brandon H. Le

doi:10.21769/BioProtoc.3680

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Peer-reviewed

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 email

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: 5416

评审: Zhibing LaiAntony ChettoorJason Liang Pin Ng

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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)

RNA Degradation (RNA降解)

Arabidopsis (拟南芥)

RT-qPCR (RT-qPCR)

Cordycepin (虫草素)

Primary MicroRNA Transcripts (初级MicroRNA转录本)

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

Supplies
1. Petri dishes, polystyrene, size 150 mm × 15 mm, sterile
2. 6-well clear multiwell plates, sterile
3. Kimwipes^TM
4. Low retention tips
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).
Reagents
1. Liquid nitrogen
2. RNaseZap^TM (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 (SYBR^TM 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 SYBR^TM 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

Multichannel pipettes
Mortar and pestle
Autoclave
-20 °C freezer
Arabidopsis growth room or chamber
VWR^® Standard Orbital Shaker, Model 3500 (VWR, catalog number: 89032-092 )
Eppendorf Benchtop Centrifuge 5418, non-refrigerated, with Rotor FA-45-18-11 and rotor lid, 230 V/50-60 Hz (Eppendorf, catalog number: 5418000017 )
Applied Biosystems^TM QuantStudio^TM 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

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

Procedure

English

中文翻译

文章信息

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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.