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Improved method for isolation of high-quality total RNA from Agave tequilana Weber roots.

3 Biotech (2021-01-29)
Luis F Maceda-López, José L Villalpando-Aguilar, Eleazar García-Hernández, Emmanuel Ávila de Dios, Silvia B Andrade-Canto, Dalia C Morán-Velázquez, Lorena Rodríguez-López, Demetrio Hernández-Díaz, Manuel A Chablé-Vega, Laura Trejo, Elsa Góngora-Castillo, Itzel López-Rosas, June Simpson, Fulgencio Alatorre-Cobos
ABSTRACT

Together with their undeniable role in the ecology of arid and semiarid ecosystems, Agave species are emerging as a model to dissect the relationships between crassulacean acid metabolism and high efficiency of light and water use, and as an energy crop for bioethanol production. Transcriptome resources from economically valuable Agaves species, such as Agave tequilana and A. salmiana, as well as hybrids for fibers, are now available, and multiple gene expression landscape analyses have been reported. Key components in molecular mechanisms underlying drought tolerance could be uncovered by analyzing gene expression patterns of roots. This study describes an efficient protocol for high-quality total RNA isolation from phenolic compounds-rich Agave roots. Our methodology involves suitable root handling and collecting in the field and using saving-time commercial kits available. RNA isolated from roots free of lignified out-layers and clean cortex showed high values of quality and integrity according to electrophoresis and microfluidics-based platform. Synthesis of long full-length cDNAs and PCR amplification tested the suitability for downstream applications of extracted RNA. The protocol was applied successfully to A. tequilana roots but can be used for other Agave species that also develop lignified epidermis/exodermis in roots.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
TRI Reagent®, For processing tissues, cells cultured in monolayer or cell pellets
Sigma-Aldrich
Basic Fuchsin, Dye content >85 %
Sigma-Aldrich
Spectrum Plant Total RNA Kit, sufficient for 10 purifications