miR-mimic transfected cells was evaluated after the transfection by qRT-PCR. There was a significant difference between mimic transfected cells ( miR Ct value = 14,54 and miR negative control Ct value = 26,16). By mRNA microarray, most of the interested genes were found up-regulated...!!
By miR-mimic do you mean a control miR? Are you using a plasmid to express the miRs or directly transfecting the miRs?
Transfecting cells will definitely change expression of a number of genes, that is why using appropriate transfection controls are so important. Did your housekeeping gene change a lot too? If your housekeeping gene has decreased a lot, it could indicate toxicity (less cells) due to the transfection itself.
What will likely be important in your experiment are the genes in the miR cells compared to the miR control cells. Those changes should be the ones that may be due to the activity of your specific miR, and not a side effect of the transfection itself.
Dear Dr. Finn : miR-X mimic was transfected into cell line directly using Hiperfect Qiagen transfection reagent, and as a control, I used siRNA negative control and un-transfected cells. Therefore, in this case we have 9 wells (3 wells for miR-X mimic, 3 wells for siRNA negative control and 3 wells for only cells or un-transfected cells). Three days after transfection, the expression of miR-X along with siRNA and un-transfected cells has been evaluated by qRT-PCR. The cells/wells, which have been transfected with miR-X mimic, were up-regulated or the Ct value was 14 and 26 for the siRNA and un-transfected cells/wells. After that, I isolated the RNA for mRNA microarray from each well separately. After miR-X mimic transfection, most of the genes were up-regulated and only few number of genes were down regulated, even the genes that have a role in my topic, were up-regulated. I validated the mRNA microarray results by qRT-PCR for some selected genes, but I also found same results as in the mRNA microarray (genes were found up regulated).
Hi Masood,
So I am still not clear, are you transfecting plasmids expressing the miR-X and siRNA, or directly transfecting the miR-X and siRNA molecules? Why are you using siRNA as a control for the miR-X? siRNA and miRs have different structures, why not control for your miR-X with a miR-scramble?
I assume that the miR-X was used to downregulate expression of a gene (or group of genes?). Did you confirm that you were getting downregulation of your target gene?
How did the cells look after transduction? Did all conditions look the same? Did the cells look normal and healthy?
J
Hello Dr. Finn
I have directly transfected the cells with both miR-X and siRNA separately. siRNAs as a control because siRNA or miR-control can be used as control according to studies and also according to Qiagen recommendations,
There was no effect by qRT-PCR between siRNA and un-transfected cells. After transfection, genes were found up-regulated by both mRNA microarray and qRT-PCR for some selected genes.? But, there was a significant effect between miR-x and siRNA and/or un-transfected cells. I have mentioned this in my above listed post.
the level of apoptosis by FACS for mimic transfected cells was increased compared to controls and even the morphology was changed in mimic transfected cells... the condition does not look same between miR-x transfected cells and normal cells or cells transfected with siRNA.?
Perhaps the miRNA used for transfection is silencing inhibitory factors of the genes found up-regulated.
Hi Neha,
Transfection of small RNAs like miRNAs into cells typically lowers expression of many genes and also could increase the expression of certain target genes. It could be resulted from the competition between the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. I think you do not need to run microarry!
have a look at
Nature Biotechnology 27, 549 - 555 (2009)
Masood
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