Abstract
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.
Highlights
Plant sedentary endoparasitic nematodes from Meloidogyne spp. genus, represent a serious threat to the agricultural production (McCarter, 2009)
We present a protocol that allows the simultaneous extraction of DNA and total RNA with high quality and enough yield for high throughput sequencing purposes (Figure 1) from the same biological sample of M. javanica-induced galls and its respective control root segments
An emerging topic coming from the molecular analyses combining transcriptomics with cell biology, such as laser microdissection of giant cells of different plant species, is that early developing GCs are characterized by widespread, but specific, gene repression that includes many microRNAs (Barcala et al, 2010; Portillo et al, 2013; Cabrera et al, 2016; Medina et al, 2017)
Summary
Plant sedentary endoparasitic nematodes from Meloidogyne spp. genus (root-knot nematodes), represent a serious threat to the agricultural production (McCarter, 2009). We present a protocol that allows a simultaneous extraction and purification of genomic DNA and total RNA from the same plant tissue sample, focused on feeding structures formed by a plant-parasitic nematode, Meloidogyne javanica, in Arabidopsis, and its respective control roots. For RNA extraction, the customized protocol proposed by the manufacturer is followed using the upper aqueous phase obtained, after adding 90 μL of chloroform to the flow-through of the AllPrep DNA Mini spin column (QIAGEN ; see former section). To elute the RNA, 30 μL RNase-free water is added directly to the spin column membrane and centrifuged for 1 min at 9000 × g and this step is repeated at least a second time using a new microcentrifuge tube (15) RNase-Free DNase Set [with Buffer RDD, included in the AllPrep DNA/RNA/miRNA Universal Kit, QIAGEN , 1500 Kunitz units, 50 reactions It is important to check the remaining volume from time to time
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