My lab is transfecting HL-60 cells with siRNA targeting PLAC8 using lipid based transfection (lipofectamine). We have tried various concentrations for both the cells and siRNA (10, 30, 60 and 90 nm siRNA in 2 mL of cells)(cells ranging from 1 E5 to 1 E6 cells/mL) in accordance with many different protocols and methods from lit searches.
We have consistently gotten very low RNA yields following the transfection, but increasing the concentration of cells has led to loss of any effect on the expression of the target gene. Furthermore, the control gene changes in expression with a pattern similar to the target gene.
I think we need to increase the concentration of cells, and in that way hopefully get higher RNA yields and fewer universal silencing effects, but is there any way to increase efficiency of the transfection so that increasing cell concentration will not lead to the loss of any specific PLAC8 downregulation?
We are considering switching to plasmid-based transfection, but due to the increased cost and the nature of our research (many genes of interest), we would much prefer to stick with directly transfecting shRNA.