Polysome Analysis
多聚核糖体分析
发布: 2017年03月20日第7卷第6期 DOI: 10.21769/BioProtoc.2192 浏览次数: 22640
评审: HongLok LungAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2016
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Abstract
Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription – PCR to investigate the translational state of the mRNA.
Keywords: Translation (翻译)
Ribosome fractionation (核糖体分离)
Polysome (多聚核糖体)
mRNA (mRNA)
Sucrose density gradient (蔗糖密度梯度)
Ultracentrifugation (超速离心)
Background
The cellular mRNAs are distributed into an actively translating and a non-translating pool at any point of time and can dynamically redistribute between these pools in response to various stimuli. The actively translating mRNAs have a higher number of ribosomes associated with them and the number of ribosome associated with an mRNA is a measure of the translation state of the mRNA. Therefore on fractionating the ribosomes from a cell, actively translating mRNAs will be found in the polysomal fraction whereas non-translating/poorly-translating mRNAs will be either found in the free mRNA fraction or associated with 40S ribosomal subunits. Polysome analysis is therefore a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes (Ray et al., 2009; Poria et al., 2016). Association of individual mRNAs with translating/non-translating fractions can be detected by RT-PCR, whereas the entire translation or non-translating pool of mRNAs can be identified by RNA sequencing or microarray analysis.
Materials and Reagents
- Ultracentrifuge tube for SW41Ti (Seton Scientific, catalog number: 7030 )
- Permanent ink marker
- 10 ml syringe with wide gauge luer lock cannula (Vita Needles, gauge 14)
- 10 cm dish (Eppendorf, catalog number: 0030702115 )
- 1.5 ml tube (RNase and DNase free) (Corning, Axygen®, catalog number: MCT-150-C or equivalent)
- MCF7 human breast carcinoma cell line (ATCC, catalog number: HTB22 )
- Cycloheximide (CHX) (AMRESCO, catalog number: 94271 )
- Phosphate buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010023 )
- DEPC treated water (AMRESCO, catalog number: E174 )
- Citrate saturated phenol (pH 4.5) (Sigma-Aldrich, catalog number: P4682 )
- Chloroform (Sigma-Aldrich, catalog number: C2432 )
- 75% ethanol
- Nuclease free water
- Oligo dT primer
- dNTP mix
- 5x RT buffer
- Dithiothreitol (DTT) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0861 )
- RNase inhibitor (Thermo Fisher Scientific, catalog number: EO0381 )
- cDNA synthesis reagents (Thermo Fisher Scientific, InvitrogenTM, catalog number: 28025013 )
- 10x PCR buffer (with 15 mM MgCl2)
- 10 µM forward primer for gene of interest/control gene
- 10 µM reverse primer for gene of interest/control gene
- PCR reagents (New England Biolabs, catalog number: M0273L )
- Sucrose (RNase and DNase free) (AMRESCO, catalog number: 0335 )
- Potassium chloride (KCl) (AMRESCO, catalog number: 0395 )
- Magnesium chloride (MgCl2) (AMRESCO, catalog number: 0288 )
- HEPES (pH 7.4) (Thermo Fisher Scientific, Affymetrix, catalog number: 16926 )
- IGEPAL CA-360 detergent (Sigma-Aldrich, catalog number: I3021 )
- Protease inhibitor cocktail (AMRESCO, catalog number: M250 )
- 10% sucrose solution (for 8 ml) (see Recipes)
- 50% sucrose solution (for 8 ml) (see Recipes)
- Polysome lysis buffer (see Recipes)
Equipment
- Gradient Station or any equivalent gradient fractionators (Biocomp, catalog number: 153-002 )
- Tube holder
- Ultra-centrifuge (Beckman Coulter, model: Optima L-70 or equivalent)
- Rotor SW41Ti (Beckman Coulter, catalog number: 331362 )
- 200 µl pipette
- Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000 )
- Fraction collector (Gilson, model: FC203B )
- UV monitor (Bio-Rad Laboratories, model: EM1-EconoTM )
- Balance
- Vortex
Software
- Gradient Master software (included with the Gradient Station)
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Poria, D. K. and Ray, P. S. (2017). Polysome Analysis. Bio-protocol 7(6): e2192. DOI: 10.21769/BioProtoc.2192.
Download Citation in RIS Format
分类
癌症生物学 > 癌症生物化学 > 蛋白质
生物化学 > RNA > RNA-蛋白质相互作用
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