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, Sucrose-gradient seperation, In vivo translation of genes
Materials and Reagents
Equipment
Procedure
Pre-treatment: All glass materials were heated in 180 °C for 12 h and all plastic materials were treated with non-sterile DEPC-H2O overnight, then autoclaved at 121 °C for 20 min. RNase-free water was used to prepare all buffers.
Representative data
Figure 1. Polysomal profiles and validation of the specificity of immunopurification of mRNAs associated with pollen from pollinated floral buds, in vivo-pollinated pollen tubes, and in vitro-cultured pollen tubes of LAT52:HF-RPL18 transgenic plants. (A) Typical sucrose-gradient absorbance (A 260) profiles of ribosome complexes obtained from Bud stage, in vivo stage, and in vitro-cultured pollen. Positions of peaks corresponding to polysomes (line), 40S ribosomal subunits and 60S ribosomal subunit/80S monosomes (arrowheads) are indicated. The arrow for the sedimentation reflects the sucrose gradient from 20% to 60% (top to bottom). (B) Quantitative RT-PCR with primer sets targeting the FLAG-RPL18 transgene and organ-specific genes to confirm that the RNA extracted from purified polysomal mRNAs examined by sucrose-gradient separation and further purified by FLAG-agarose beads was all male gametophyte-specific. Primers span both the His6-FLAG tag and the RPL18 sequence (FLAG-PRL18); pollen-specific PLIM2 and VGD1, female-specific SHP1 and SR, and petal/sepal-specific AP1. ACT2 was an internal control (Lin et al., 2014). Table1. Primer pairs for Q-PCR in Figure 1 (Lin et al., 2014)
Notes
Recipes
Acknowledgments
We thank Dr. Julia Bailley-Serres (Department of Botany and Plant Sciences, University of California, Riverside) for the pLAT52-HF-RPL18 Arabidopsis and for sharing the polysomal-mRNA extraction protocol. This work was funded by Academia Sinica (Taiwan), the Taiwan National Science and Technology Program for Agricultural Biotechnology (Lin et al 31; NSTP/AB, 098S0030055-AA) and the Taiwan National Science Council (99-2321-B-001-036-MY3 and 102-2321-B-001-040-MY3) to G.-Y. Jauh.
References
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