A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissue-specific expression profiling of rice roots
| Autor: | Mounier, Thibault, Navarro-Sanz, Sergi, Bureau, Charlotte, Antoine, Lefeuvre, Varoquaux, Fabrice, Durandet, Franz, Périn, Christophe |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
F60 - Physiologie et biochimie végétale
ddRT-PCR Coiffe Oryza sativa Laser Capture Microdissection Plant Roots F50 - Anatomie et morphologie des plantes Tissu végétal F30 - Génétique et amélioration des plantes Gene Expression Regulation Plant gene expression profiling [EN] lcsh:QH573-671 Paraffin Embedding Reverse Transcriptase Polymerase Chain Reaction lcsh:Cytology Methodology Article Gene Expression Profiling Reproducibility of Results Oryza High-Throughput Screening Assays Laser microdissection (LM) Droplet digital PCR Organ Specificity RNA Plant Cortex Méristème Rice Root meristem Racine |
| Zdroj: | BMC Molecular and Cell Biology, Vol 21, Iss 1, Pp 1-17 (2020) BMC Molecular and Cell Biology |
| ISSN: | 2661-8850 |
| Popis: | Background In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. Results We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. Conclusion The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|