Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses

• Autores: Avico, Edgardo Hernán; Acevedo, Raúl Maximiliano; Ruiz, Oscar Adolfo; Sansberro, Pedro Alfonso; Calzadilla, Pablo I.
• Año de publicación: 2017
• Idioma: inglés
• Tipo de recurso: artículo
• Fuente: Plant Omics Journal, 2007, vol. 10, no. 4, p. 205-209.
• Materia: Clay soi; Dehydration; Llex paraguariensis; RNA isolation; RNA-seq; Root tissues; Woody plant
• Institución: Universidad Nacional del Nordeste

Fil: Avico, Edgardo Hernán. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina.
Fil: Acevedo, Raúl Maximiliano. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina.
Fil: Acevedo, Raúl Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina.
Fil: Ruiz, Oscar Adolfo. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas ; Argentina.
Fil: Ruiz, Oscar Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Biotecnológicas; Argentina.
Fil: Sansberro, Pedro Alfonso. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias; Argentina.
Fil: Sansberro, Pedro Alfonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Botánica del Nordeste; Argentina
Fil: Calzadilla, Pablo I. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico La Plata. Instituto de Investigaciones Biotecnológicas; Argentina.
Fil: Calzadilla, Pablo I. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina.
Extraction of high quality RNA is a prerequisite for downstream application in functional genomics analyses. However, the extraction and purification of pure nucleic acids from root tissues is generally difficult due to the high concentration of carbohydrates and secondary metabolites. Furthermore, the presence of enzymatic inhibitors such as fulvic and humic acids can also negatively affect extraction quality, when extracting from clay soil-grown roots. In this work, total RNA was extracted from soil-grown roots of Ilex paraguariensis using four commercially available kits: SpectrumTM, RNeasy®, TRI Reagent®, and SV Total RNA Isolation System. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The SpectrumTM and RNeasy® protocols provided the highest quantity and quality of RNA; however, the former revealed superior extraction performance. Consequently, total RNA was extracted from the roots of non-stressed and drought-stressed plants using the SpectrumTM method and six RNA-seq libraries were prepared from polyA + mRNAs by means of TruSeq mRNA library construction protocol to convert RNA to complementary DNA (cDNA). More than 80 million raw read sequences were obtained from each condition with an average read length of 150 bp. The yield and quality of the total RNA were consistently high and the RNA could be used for further analyses as demonstrated by cDNA library construction, RT-PCR, and transcriptome sequencing. Thus, SpectrumTM method can be used to isolate high quality RNA from roots of normal and drought stressed I. paraguariensis plants.