I found that over-expressed RGL3 can induce cell migration. I bought siRNA RGL3 and transfected to NIH3T3 cell line. Then, I observe cell migration of depleted cell but I could not observe a decrease in cell migration. My interpretation that these protein have redundant function so if we knock down only RGL3, we could not observe a defect in cell migration.
I intend to generate siRNA that can block all the family of RGL (RGL1, RGL2, and RGL3). Is it a good idea? Is it scientific sense?
Or I have to generate seperate siRNA RGL1, RGL2, and RGL3, then transfect these 3 siRNA in one cell line to make sure all of protein in RGL family is completely delepted.
As Jonathan indicates, 3 separate siRNAs are probably the best. The attached sequence alignment of Mus musculus Rgl1, Rgl2, and Rgl3 shows a couple of regions with fairly good identity, but there really is no reason to use just one siRNA.
Hello Nguyen,
I have never worked with the RGL family so I don't know how similar the sequences are of RGL1, RGL2 and RGL3. If you do a sequence alignment and find some regions that are identical, then in theory it might be possible to knock down all three genes with one siRNA, however you will have to verify this as all siRNA target sites are not equal when it comes to knocking down activity. Using 3 different siRNA is probably the better way to go, but you will have to make sure that each siRNA really does knock down your gene.
Are you confirming knockdown by western blot?
Good luck
As Jonathan indicates, 3 separate siRNAs are probably the best. The attached sequence alignment of Mus musculus Rgl1, Rgl2, and Rgl3 shows a couple of regions with fairly good identity, but there really is no reason to use just one siRNA.
@Johnathan I could not detect the endogenous band for RGL3. I only confirm siRNA RGL3 by siRNA.
@Mark: Thanks for alignment
Johnathan and Mark: do you know any program that I can use to align the sequence and find the identical region of different family of a gene? I know BioEdit, Snapgene, and Edit sequence. However, I dont know how to know which region are idential or similar and the percentage of similarity.
@Mark: I looked at the sequence but I dont know in which regions they are identical. I dont have experience about this.
I use CloneManager, but that program isn't free. You can also use online programs like Clustal Omega. By lining up all three genes you should be able to look for regions of homology (exactly like Mark did using the mouse genes).
@Mark: I look at the pdf file you send me. I think that the identical region you mentioned is the region that has many asterisks. From the attached alignment , I found 3 regions that have many asterisks.Thank you, I will design siRNA based on these identical regions.
@All: Can I ask one more question,?how can we know the percentage of identical? The software can do it for us? Can I know which software it is?
Here is link for software to design siRNA. U need to design it for each gene separately.
http://www.sirnawizard.com/siRNA.php
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