A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissue-specific expression profiling of rice roots

Mounier Thibault, Navarro Sanz Sergi, Bureau Charlotte, Lefeuvre Antoine, Varoquaux Fabrice, Durandet Franz, Périn Christophe. 2020. A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissue-specific expression profiling of rice roots. BMC Molecular and Cell Biology, 21:92, 17 p.

Url - jeu de données - Entrepôt autre : https://figshare.com/articles/presentation/Additional_file_1_of_A_fast_efficient_and_high-throughput_procedure_involving_laser_microdissection_and_RT_droplet_digital_PCR_for_tissue-specific_expression_profiling_of_rice_roots/13364202

Quartile : Q4, Sujet : CELL BIOLOGY

Résumé : 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.

Mots-clés Agrovoc : Oryza sativa, racine, profilage de l'expression génique, tissu végétal, cortex, méristème, coiffe

Mots-clés complémentaires : microdissection laser, RT-PCR, Aérenchyme

Mots-clés libres : Rice, Root anatomy, Laser Microdissection, RT-ddPCR

Classification Agris : F50 - Anatomie et morphologie des plantes
F30 - Génétique et amélioration des plantes
F60 - Physiologie et biochimie végétale

Champ stratégique Cirad : CTS 2 (2019-) - Transitions agroécologiques

Auteurs et affiliations

• Mounier Thibault, CIRAD-BIOS-UMR AGAP (FRA)
• Navarro Sanz Sergi, CIRAD-BIOS-UMR AGAP (FRA)
• Bureau Charlotte, Université de Montpellier (FRA)
• Lefeuvre Antoine, IAGE Company (FRA)
• Varoquaux Fabrice, Université de Montpellier (FRA)
• Durandet Franz, IAGE Company (FRA)
• Périn Christophe, CIRAD-BIOS-UMR AGAP (FRA) ORCID: 0000-0002-2469-310X - auteur correspondant

Source : Cirad-Agritrop (https://agritrop.cirad.fr/597240/)

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