TY  - JOUR
TI  - Polysomal-mRNA Extraction from Arabidopsis by Sucrose-gradient Separation
AU  - Lin, Shih-Yun 
AU  - Jauh, Guang-Yuh 
VL  - 4
IS  - 24
PY  - 2014
DA  - 2014/12/20
SP  - e1364
C1  - Bio-protocol 2014;4:e1364
DO  - 10.21769/BioProtoc.1364
UR  - https://doi.org/10.21769/BioProtoc.1364
AB  - 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.
KW  - Polysomal-mRNA
KW  - Sucrose-gradient seperation
KW  - In vivo  translation of genes
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False