sequence selection for dsRNA generation for RNAi

2 posts / 0 new
Last post
nucleo77
nucleo77's picture
sequence selection for dsRNA generation for RNAi

I am doing a study regarding an dsRNA mediated interference RNA in plants (tomato) using 2 fused genes that would target delayed ripening and viral resistance, however,i see most programs to generate either miRNA or shiRNA. are there any algoritms developed to generate the cDNA targets for dsRNA initiation of RNAi?

Second, instead of a vector based RNAi i'm thinking of using synthetic oligonucleotides (100mers) to be ligated into the vector with my choice intron. My challenge is, I would order my oligos as a very long primer therefore it is single stranded, but my desire is to have a cDNA with both strands so i can produce the antisense by using directional primers designed with specific restriction sites. What should I do with my synthetic 100mer oligonucleotide prior to PCR to produce the double strands?

Thirdly, is there an in vitro screening technique developed to evaluate RNAi for plant systems?

behrooz
behrooz's picture
Hi,nucleo77

Hi,nucleo77
you can use RT-PCR for amplification of favorite fragment with specific primers at desire region of mRNA. also for studing of transformants you can appliy real-time PCR TO detection of transcript varities or do two-dimentional electrophoresis for detection of protein changs. also, you can do ELISA with specific antibody.
behrooz