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

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,...

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Autores principales: Avico, Edgardo Hernán, Acevedo, Raúl Maximiliano, Ruiz, Oscar Adolfo, Sansberro, Pedro Alfonso, Calzadilla, Pablo I.
Formato: Artículo
Lenguaje:Inglés
Publicado: Southerm Cross 2021
Materias:
Clay soi
Dehydration
Llex paraguariensis
RNA isolation
RNA-seq
Root tissues
Woody plant
Acceso en línea:http://repositorio.unne.edu.ar/handle/123456789/27780
Aporte de:
RIUNNE - Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE) de Universidad Nacional del Nordeste
id I48-R184-123456789-27780
record_format dspace
spelling I48-R184-123456789-277802024-10-09T14:22:18Z Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses Avico, Edgardo Hernán Acevedo, Raúl Maximiliano Ruiz, Oscar Adolfo Sansberro, Pedro Alfonso Calzadilla, Pablo I. Clay soi Dehydration Llex paraguariensis RNA isolation RNA-seq Root tissues Woody plant 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. 2021-04-13T17:19:43Z 2021-04-13T17:19:43Z 2017-07 Artículo Avico, Edgardo Hernán, et al., 2017. Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses. Plants Omics Journal. Australia: Southerm Cross, vol. 10, no. 4, p. 205-209. ISSN1836-3644. 1836-0661 http://repositorio.unne.edu.ar/handle/123456789/27780 eng https://www.pomics.com/sanberro_10_4_2017_205_209.pdf openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ application/pdf application/pdf Southerm Cross Plant Omics Journal, 2007, vol. 10, no. 4, p. 205-209.
institution Universidad Nacional del Nordeste
institution_str I-48
repository_str R-184
collection RIUNNE - Repositorio Institucional de la Universidad Nacional del Nordeste (UNNE)
language Inglés
topic Clay soi
Dehydration
Llex paraguariensis
RNA isolation
RNA-seq
Root tissues
Woody plant
spellingShingle Clay soi
Dehydration
Llex paraguariensis
RNA isolation
RNA-seq
Root tissues
Woody plant
Avico, Edgardo Hernán
Acevedo, Raúl Maximiliano
Ruiz, Oscar Adolfo
Sansberro, Pedro Alfonso
Calzadilla, Pablo I.
Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses
topic_facet Clay soi
Dehydration
Llex paraguariensis
RNA isolation
RNA-seq
Root tissues
Woody plant
description 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.
format Artículo
author Avico, Edgardo Hernán
Acevedo, Raúl Maximiliano
Ruiz, Oscar Adolfo
Sansberro, Pedro Alfonso
Calzadilla, Pablo I.
author_facet Avico, Edgardo Hernán
Acevedo, Raúl Maximiliano
Ruiz, Oscar Adolfo
Sansberro, Pedro Alfonso
Calzadilla, Pablo I.
author_sort Avico, Edgardo Hernán
title Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses
title_short Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses
title_full Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses
title_fullStr Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses
title_full_unstemmed Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses
title_sort isolation of high quality rna from soil-grown ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses
publisher Southerm Cross
publishDate 2021
url http://repositorio.unne.edu.ar/handle/123456789/27780
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