I am trying to suppress Long Non Coding RNAs expression in cultured mice splenocytes. I'm using a pooled siRNA composed of 4 siRNAs. The method of transfection is based on lipofectamin and after 4 hours of transfection, stimulation with anti-CD3/CD28 is done. Unfortunately after 24 hours of transfection I was not able to detect downregulated expression of target genes. Are there any details I may have missed here?
Detecting endogenous genes is tricky, usually lacks sensitivity and if working with primary cells is more challenging. I'd suggest cotransfecting a reporter plasmid containing your target sequence coded inframe or driven by an independent IRES from a reporter such as Luciferase or Green Fluorescent Protein in the same cistron. You can assess successful downregulation of your target by measuring the reporter either by a quantitative assay or by microscopy. I followed such strategy to knockdown a huge gene. Please see http://www.ncbi.nlm.nih.gov/pubmed/15131132 . Good luck
Detecting endogenous genes is tricky, usually lacks sensitivity and if working with primary cells is more challenging. I'd suggest cotransfecting a reporter plasmid containing your target sequence coded inframe or driven by an independent IRES from a reporter such as Luciferase or Green Fluorescent Protein in the same cistron. You can assess successful downregulation of your target by measuring the reporter either by a quantitative assay or by microscopy. I followed such strategy to knockdown a huge gene. Please see http://www.ncbi.nlm.nih.gov/pubmed/15131132 . Good luck
Hello
i agree with Oscar l., please look at the links here may help you
Good Luck
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583990/
http://www.science.gov/topicpages/s/sirna+mediated+mrna.html
http://www.sciencedirect.com/science/article/pii/S221128391300004X
https://docs.google.com/viewer?url=patentimages.storage.googleapis.com/pdfs/US20140056929.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418769/
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