采用甘蔗梯度分离法提取拟南芥多聚核糖体-mRNA

Shih-Yun Lin; Guang-Yuh Jauh

doi:10.21769/BioProtoc.1364

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Peer-reviewed

Polysomal-mRNA Extraction from Arabidopsis by Sucrose-gradient Separation

采用甘蔗梯度分离法提取拟南芥多聚核糖体-mRNA

SL Shih-Yun Lin

Guang-Yuh Jauh email

发布: 2014年12月20日第4卷第24期 DOI: 10.21769/BioProtoc.1364 浏览次数: 12260

评审: Tie LiuAnonymous reviewer(s)

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Feb 2014

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Abstract

mRNAs surrounded by polysomes are ready for translation into proteins (Warner et al., 1963); these mRNAs are defined as polysomal-mRNAs (Mustroph et al., 2009). The process is affected by various growth conditions or surrounding situations. Microarray analysis is a powerful tool for detecting genome-wide gene expression. Therefore, using polysomal-mRNAs for microarray analysis can reflect the gene translation information (the translatome) under different developmental stages or environmental conditions from eukaryotes. Polysomal-mRNAs can be collected from the polysomal fraction by sucrose-gradient separation for further quantitative PCR or microarray assay. We modified a protocol (Mustroph et al., 2009) for collecting polysomal-mRNAs via sucrose-gradient separation to eliminate monosomal-mRNA contamination from pLAT52:HF:RPL18 Arabidopsis. This transgenic Arabidopsis uses a pollen-specific promoter (ProLAT52) to generate epitope-tagged polysomal-RNA complexes that could be purified with a specific antigen (Lin et al., 2014). The polysomal-mRNAs we obtained via sucrose-gradient separation and antibody purification underwent in vivo translation in pollen tubes grown from self-pollinated gynoecia of Arabidopsis thaliana.

Keywords: Polysomal-mRNA (多聚核糖体的mRNA)

Sucrose-gradient seperation (蔗糖密度梯度分离)

In vivo translation of genes (基因的体内翻译)

Materials and Reagents

pLAT52: HF-RPL18 transgenic Arabidopsis
RNase-free water
Tris buffer (Sigma-Aldrich, catalog number: T1378 )
KCl (Sigma-Aldrich, catalog number: P9541 )
EGTA (Sigma-Aldrich, catalog number: E3889 )
MgCl₂ (Sigma-Aldrich, catalog number: M8266 )
β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
Cycloheximide (Sigma-Aldrich, catalog number: C7698 )
Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
Polyoxyethylene 10 tridecyl ether (PTE) (Sigma-Aldrich, catalog number: P2393 )
Sodium deoxycholate (DOC) (Sigma-Aldrich, catalog number: D6750 )
Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
Triton X-100 (Sigma-Aldrich, catalog number: X-100 )
Polyoxyethylene(23)lauryl ether (Brij -35) (Sigma-Aldrich, catalog number: P6938 )
Tween-20 (Sigma-Aldrich, catalog number: P1379 )
NP-40 (Sigma-Aldrich, catalog number: NP 40 )
Polyoxyethylene (Sigma-Aldrich, catalog number: P2393)
Deoxycholic acid (Sigma-Aldrich, catalog number: D2510 )
ANTI-FLAG^® M2 Affinity Gel (Sigma-Aldrich, catalog number: A2220 )
RNAsin (Promega Corporation, catalog number: N2511 )
3x FLAG peptide (Sigma-Aldrich, catalog number: F4799 )
Sucrose (Sigma-Aldrich, catalog number: 84097 )
Polysomal extraction buffer preparation (see Recipes)
20% detergent mixture (see Recipes)
20% PTE and 10% DOC (see Recipes)
Sucrose gradient layer preparation (see Recipes)
10x sucrose salts (see Recipes)
Preparation of the FLAG M2 Agarose beads (see Recipes)

Equipment

QIAGEN QIA shedder columns (QIAGEN, catalog number: 27115 )
15-ml tube (Labcon, catalog number: 9205-946CB-946C )
Hitachi ultracentrifuge himac CP100WX (Hitachi, catalog number: CP100WX )
Hitachi P40ST swing rotor (Hitachi, model: P40ST )
13PA tubes (Hitachi, catalog number: 332901A )
Spectrophotometer (Beckman Coulter, catalog number: DU648B )
UV monitor (GE Healthcare, catalog number: UVIS-920 )
Pump (GE Healthcare, model: P-50 )
Fraction collector (Gilson Products, catalog number: FC80 )
UV detector system (GE Healthcare, catalog number: monitor UVIS-0912 )
Recorder (GE Healthcare, catalog number: pharmaria LKB REC102 )

Procedure

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Lin, S. and Jauh, G. (2014). Polysomal-mRNA Extraction from Arabidopsis by Sucrose-gradient Separation . Bio-protocol 4(24): e1364. DOI: 10.21769/BioProtoc.1364.