RNA silencing is a fundamental plant defence and gene control mechanism in plants that are directed by 20-24 nt small interfering RNA (siRNA) and microRNA (miRNA). RNA silencing has been exploited to develop powerful gene silencing technologies such as the hairpin RNA (hpRNA) transgene technology. Since siRNAs are processed from long dsRNA by Dicer-like proteins, the expected ratio between sense and antisense siRNA strands should be 1:1. However, siRNAs from either the plant genome (e.g. hpRNA transgene) or from infecting viruses often show a strong unequal distribution of the sense and antisense strands. How the strand bias occurs in plants remains unclear, although studies in animal cells have implicated an involvement of target RNA in affecting small RNA accumulation. To investigate this, we used Arabidopsis and tobacco as model plants to examine the accumulation of hpRNA transgene-derived sRNA in the presence or absence of target mRNA. Results from transient expression using Agrobacterium infiltration showed that the presence of abundant target RNA affects the accumulation of anti-sense siRNA. To examine the effect of target RNA on siRNAs from stably integrated hpRNA transgenes, we generated plants expressing hpRNA alone or both hpRNA and target mRNA. These plants are being analysed for siRNA abundance, and results will be presented. Since siRNA polarity (sense or antisense), sequence composition and abundance are important for the ability of the siRNA to induce efficient silencing, understanding the factors that affect the siRNA abundance can potentially be used to improve the efficiency of gene silencing in plants.