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.
Department of Plant Systems Biology, VIB, Ghent University, Ghent, Belgium.
Leen
L
Vermeersch
Nancy
N
De Winne
Ann
A
Depicker
D060054:Agrobacterium / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification; D035264:Flowers / Q000235:genetics; D005470:Fluorometry; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics; D056426:Genetic Loci / Q000235:genetics; D005821:Genetic Techniques; D006824:Hybridization, Genetic; D018515:Plant Leaves / Q000235:genetics; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics; D014170:Transformation, Genetic
D029681:Arabidopsis Proteins; D034741:RNA, Small Interfering