Detection and investigation of transitive gene silencing in plants.

RNA-induced post-transcriptional silencing is a common tool in functional gene analysis and its application in crop improvement is widely investigated. However, its specificity might be impaired by off-target silencing as a result of transitivity. Generally transitivity is investigated by the detection of secondary siRNAs; however, these tests fail to demonstrate the siRNA's bioactivity. Here, we describe protocols to detect the occurrence of transitive silencing across an endogene by using a reporter GUS gene. In addition, we provide a setup to test the influence of a sequence of interest present in the primary target on the progression of transitivity.