The presence of high-molecular-weight viral RNAs interferes with the detection of viral small RNAs
Abstract
Viral small interfering RNA (siRNA) accumulation in plants is reported to exhibit a strong strand polarity bias, with plus (+) strand siRNAs dominating over minus (−) strand populations. This is of particular interest, as siRNAs processed from double-stranded RNA would be expected to accumulate equivalent amounts of both species. Here, we show that, as reported, (−) strand viral siRNAs are detected at much lower levels than (+) strand-derived species using standard Northern hybridization approaches. However, when total RNA is spiked with in vitro-transcribed antisense viral genomic RNA, (−) strand viral siRNAs are detected at increased levels equivalent to those of (+) strand siRNA. Our results suggest that (+) and (−) strand viral siRNAs accumulate to equivalent levels; however, a proportion of the (−) strand siRNAs are sequestered from the total detectable small RNA population during gel electrophoresis by hybridizing to the high-molecular-weight sense strand viral genomic RNA. Our findings provide a plausible explanation for the observed strand bias of viral siRNA accumulation, and could have wider implications in the analysis of both viral and nonviral small RNA accumulation.
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Footnotes
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Reprint requests to: Ming-Bo Wang, CSIRO Plant Industry, Canberra ACT 260l, Australia; e-mail: ming-bo.wang{at}csiro.au; fax: 61-2-6246-5000.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.2049510.
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- Received December 14, 2009.
- Accepted February 11, 2010.
- Copyright © 2010 RNA Society