Effectiveness of RNA interference in transgenic plants.
RNA interference (RNAi) can be used to study gene function by effecting degradation of the targeted transcript. However, the effectiveness of transgene-induced RNAi among multiple target genes has not been compared systematically. To this end, we developed a relative quantitative RT-PCR protocol that allows use of a single internal standard over a wide range of target gene expression levels. Using this method in an analysis of transgenic Arabidopsis thaliana RNAi lines targeting 25 different endogenes revealed that independent, homozygous, single-copy (sc) T4 lines targeting the same gene generally reduce transcript levels to the same extent, whereas multi-copy RNAi lines differed in the degree of target reduction and never exceeded the effect of sc transgenes. The maximal reduction of target transcript levels varied among targets. These observations suggest that each target sequence possesses an inherent degree of susceptibility to dsRNA-mediated degradation.
Kerschen
Arthur
A
Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA.
Napoli
Carolyn A
CA
Jorgensen
Richard A
RA
Müller
Andreas E
AE
D017360:Arabidopsis / Q000235:genetics; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification / Q000235:genetics; D017343:Genes, Plant / Q000235:genetics; D006720:Homozygote; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D019076:Transgenes
D018076:DNA, Complementary