How to design particular SiRNA. I need detailed explanation. various websites are there for designing but how to confirm that particular SiRNA is suitable for my experiment.
Hi, you can see this link It contains important information
https://www.ncbi.nlm.nih.gov/gene/7099
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570406/
Dear Devi,
After designing specific siRNA, it is possible to check for specificity of targeting by using nucleotide blast (blastn):
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
This will give you an overview about specific targets and off-target sites for your siRNA. It is beneficial to check several sequences for specific siRNAs and also control siRNA.
At the end of the design the ultimate answer whether siRNA fits is supposed to be addressed on the experimental level. First, assess specific knock-down of your siRNAs on mRNA and protein level. Sometimes control siRNA or even transfection reagent may have off-target effect, affecting the expression of your gene of interest, therefore it is extremely important to include transfection reagent treated and untreated controls. You could at a later point to include these controls for functional studies as well to exclude possible off-target effect of control siRNA or transfection reagent on one of your functional read-outs.
I hope it helps,
Best wishes,
Julia.
Several companies provide you the putative siRNA based on computer based algorithm. siRNAs are good for transient knockdowns compared shRNA which gives a fairly stable knock down.
But to summarize. siRNA are 21 to 25 nucleotide long. Generally have AA in the start, G or C in the end, 40 to 60% GC content, contains a stem and loop structure.
Julia is correct that one has to experimentally find out which siRNA gives you the best knocked down.
At the same time, scrambled siRNA or a non-relevant siRNA like siRNA against GFP has to be put in as the control siRNA.
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