A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissue-sp
- PDF / 4,196,343 Bytes
- 17 Pages / 595.276 x 790.866 pts Page_size
- 61 Downloads / 144 Views
(2020) 21:92
BMC Molecular and Cell Biology
METHODOLOGY ARTICLE
Open Access
A fast, efficient and high-throughput procedure involving laser microdissection and RT droplet digital PCR for tissuespecific expression profiling of rice roots Thibault Mounier1†, Sergi Navarro-Sanz1†, Charlotte Bureau1†, Lefeuvre Antoine2, Fabrice Varoquaux1, Franz Durandet2 and Christophe Périn1*
Abstract 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. Keywords: Rice, Root meristem, Laser microdissection (LM), Cortex, ddRT-PCR, Droplet digital PCR
* Correspondence: [email protected] † Thibault Mounier, Sergi Navarro Sanz and Charlotte Bureau contributed equally to this work. 1 CIRAD, UMR-AGAP, Université de Montpellier, Avenue Agropolis, F-34398 Montpellier Cedex 5, France Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in th
Data Loading...
