All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes
Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both tr...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-05-01
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| Series: | Frontiers in Plant Science |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/article/10.3389/fpls.2019.00657/full |
| _version_ | 1819113362438488064 |
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| author | Ana Cláudia Silva Virginia Ruiz-Ferrer Ángela Martínez-Gómez Marta Barcala Carmen Fenoll Carolina Escobar Carolina Escobar |
| author_facet | Ana Cláudia Silva Virginia Ruiz-Ferrer Ángela Martínez-Gómez Marta Barcala Carmen Fenoll Carolina Escobar Carolina Escobar |
| author_sort | Ana Cláudia Silva |
| collection | DOAJ |
| description | Meloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting. |
| first_indexed | 2024-12-22T04:28:12Z |
| format | Article |
| id | doaj.art-5959143920814fdc9a567eba6c6fcbc0 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-22T04:28:12Z |
| publishDate | 2019-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-5959143920814fdc9a567eba6c6fcbc02022-12-21T18:39:06ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2019-05-011010.3389/fpls.2019.00657454852All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing PurposesAna Cláudia Silva0Virginia Ruiz-Ferrer1Ángela Martínez-Gómez2Marta Barcala3Carmen Fenoll4Carolina Escobar5Carolina Escobar6Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Toledo, SpainFacultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Toledo, SpainFacultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Toledo, SpainFacultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Toledo, SpainFacultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Toledo, SpainFacultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Toledo, SpainInternational Research Organization for Advanced Science and Technology (IROAST), Kumamoto University, Kumamoto, JapanMeloidogyne spp. are plant-parasitic nematodes that form a very complex pseudo-organ, called gall, which contains the giant cells (GCs) to nourish them. During the last decade, several groups have been studying the molecular processes accompanying the formation of these structures, combining both transcriptomics and cellular biology. Among others, it was confirmed that a generalized gene repression is a hallmark of early developing GCs formed by Meloidogyne javanica in Arabidopsis and tomato. One of the main mechanisms behind this gene repression involve small RNAs (sRNAs) directed gene silencing. This is supported not only by the described action of several microRNAs differentially expressed in galls, but by the differential abundance of 24-nucleotide sRNAs in early developing Arabidopsis galls, particularly those rasiRNAs which are mostly involved in RNA-directed DNA methylation. Their accumulation strongly correlates to the repression of several retrotransposons at pericentromeric regions of Arabidopsis chromosomes in early galls. However, the contribution of this global gene repression to GCs/galls formation and maintenance is still not fully understood. Further detailed studies, as the correlation between gene expression profiles and the methylation state of the chromatin in galls are essential to raise testable working hypotheses. A high quality of isolated DNA and RNA is a requirement to obtain non-biased and comprehensive results. Frequently, the isolation of DNA and RNA is performed from different samples of the same type of biological material. However, subtle differences on epigenetic processes are frequent even among independent biological replicates of the same tissue and may not correlate to those changes on the mRNA population obtained from different biological replicates. Herein, we describe a method that allows the simultaneous extraction and purification of genomic DNA and total RNA from the same biological sample adapted to our biological system. The quality of both nucleic acids from Arabidopsis galls formed by M. javanica was high and adequate to construct RNA and DNA libraries for high throughput sequencing used for transcriptomic and epigenetic studies, such as the analysis of the methylation state of the genomic DNA in galls (MethylC-seq) and RNA sequencing (RNAseq). The protocol presents guidance on the described procedure, key notes and troubleshooting.https://www.frontiersin.org/article/10.3389/fpls.2019.00657/fullArabidopsis thalianagenomic DNAMeloidogyne javanicanucleic acids integritysimultaneous RNA/DNA extractiontotal RNA |
| spellingShingle | Ana Cláudia Silva Virginia Ruiz-Ferrer Ángela Martínez-Gómez Marta Barcala Carmen Fenoll Carolina Escobar Carolina Escobar All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes Frontiers in Plant Science Arabidopsis thaliana genomic DNA Meloidogyne javanica nucleic acids integrity simultaneous RNA/DNA extraction total RNA |
| title | All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes |
| title_full | All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes |
| title_fullStr | All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes |
| title_full_unstemmed | All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes |
| title_short | All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes |
| title_sort | all in one high quality genomic dna and total rna extraction from nematode induced galls for high throughput sequencing purposes |
| topic | Arabidopsis thaliana genomic DNA Meloidogyne javanica nucleic acids integrity simultaneous RNA/DNA extraction total RNA |
| url | https://www.frontiersin.org/article/10.3389/fpls.2019.00657/full |
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