Extraction and molecular detection of viral dsRNA from different infected plants

Abstract

Extraction of viral double stranded RNA (dsRNA) from infected plants is helpful in identification of the viruses involved in infection. To date, there have been several methods developed to isolate dsRNA; however, type of the plant and virus is determinative in extraction efficiency. In this study we extracted dsRNA from different woody and herbaceous plants through a modified method which reduces the costs and time of extraction procedure. This method is based on different affinity of nucleic acids for the cellulose CF-11 in1X STE (Sodium chloride Tris EDTA) buffer containing 16 % ethanol. There is no phenol treatment or mini columns used in the isolation procedure. Extracted dsRNAs were identified by ribonuclease treatment and RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). We have applied the procedure on five different hosts representing Amaranthaceae, Vitaceae, Fabaceae and Rosaceae infected with four different viruses representing Secoviridae and Bromoviridae.

Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

“…1(A)) followed by release of second instar mealybug nymphs on it and dipping the leaf petiole in nuclease free water for 48 h. Mealybug nymphs were collected (n = 25 in 3 biological replicates) from leaves 72 h post release for isolation of total RNA using Tri-Reagent ® (Sigma-Aldrich)) followed by cDNA synthesis (First strand cDNA synthesis kit, ThermoFisher Scientific) and relative estimation of mRNA transcripts of targeted genes using RT-qPCR as described earlier. dsRNA was extracted from leaf post petiole dip (12 h) to confirm its uptake in leaf as per earlier described methodology 27 . Additionally, membrane feeding with 500 ng dsRNA per µl of artificial diet (Sucrose 30% solution, table sugar 50 mg and 1 ml yellow food dye (Ajanta Food Products Co., Solan, India) stretched between two layers of parafilm was tried in 2 nd and 3 rd instar, and female adult mealybug (Supplementary Information 4:Fig.…”

Characterization of a Novel Pathogenic Reovirus in Grasshoppers

“…The contigs with high expression levels but low homology to known reoviruses were then selected as candidate reovirus sequences. To further confirm all segments of reovirus genome, dsRNAs were isolated from the purified virus following the method described by Khabbazi et al [18]. dsRNAs were analyzed using 2% agarose gel electrophoresis and visualized in C-150 Gel Imager (Azure, Northlake, IL, USA).…”