|
|
Comparison of methods of isolating double-stranded RNA from plant tissue with a focus on viral fraction yield.
MATYÁŠOVÁ, Alena
High-throughput sequencing is one of methods used for diagnostics of plant pathogens and has advantage of unspecific unbiased detection of all nucleic acids present in a sample. Input material for HTS can be prepared using by different approaches that reflect purpose of the planned task. During viral infections of plants, viral double stranded RNAs are generated as replication intermediates or transcription products. Thus, they are often used for HTS. Nevertheless, such preparations contain large amount of plant RNAs. The work aimed for comparison of two methods of double stranded RNA enrichment - widely used cellulose chromatography and differential centrifugation with lithium chloride. Both qualitative and quantitative profiles of viral nucleic acids were estimated. An isolate HZ2 of red clover (Trifolium pratense L.) was used for the study. Previously, there were detected eight different RNA viruses with HTS-aimed analyses in the plant. To compare qualitative profile, RNA was extracted by each method, transcribed into cDNA, and specific viral fragments were amplified using PCR, followed by agarose gel electrophoresis. Quantitative profiles were analyzed using three selected viruses with single- and double-stranded RNA genomes. Their relative quantification was estimated using RT-qPCR approach. Further, normalized (to 26S rRNA as a reference) expressions were calculated using Bio-rad CFX Manager 3.1 software. All eight viruses were successfully detected using RNA material obtained by each method. After evaluating the quantification data and performing statistical tests (F-test, T-test; with a significance level of = 0.05), no significant difference was found between the compared methods.
|
guest ::
RSS feed.