Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica

Jennifer S Garbutt, Xavier Bellés, Elaine H. Richards, Stuart E. Reynolds

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi.
Original languageEnglish
Pages (from-to)171-178
Number of pages8
JournalJournal of Insect Physiology
Volume59
Issue number2
Early online date2 Jun 2012
DOIs
Publication statusPublished - Feb 2013

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Manduca
Blattella germanica
Double-Stranded RNA
Hemolymph
Manduca sexta
double-stranded RNA
RNA Interference
RNA interference
hemolymph
Insects
insects
Gene Silencing
gene silencing
Blattellidae
Reverse Genetics
nucleases
assays
Edetic Acid
Reverse Transcription
reverse transcriptase polymerase chain reaction

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Persistence of double-stranded RNA in insect hemolymph as a potential determiner of RNA interference success: Evidence from Manduca sexta and Blattella germanica. / Garbutt, Jennifer S; Bellés, Xavier; Richards, Elaine H.; Reynolds, Stuart E.

In: Journal of Insect Physiology, Vol. 59, No. 2, 02.2013, p. 171-178.

Research output: Contribution to journalArticle

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