Hi
I want to transfect 10 nM, 20 nM and 30 nM of miR145 mimic to MCF-7 cell line. Then I want to transfect a plasmid containing miR 145 target sequences at the end of my gene. I want to evaluate expression my gene versus miR 145 concentrations (10 nM, 20 nM and 30 nM).
I have two question?
1. Using miR negative control is necessary for this test?
2. It is necessary using 10 nM, 20 nM and 30 nM of miR negative control or I can use from one concentration?
Dear Mohammad,
For your experiment to be well design, you would need 1) a miR negative control, basically a scramble mimic that does not match with any sequence of the genome you are working on.
Then your transfection will be controled for transfecting an oligonucleotide in the cells.
It would be better to would also be ideal to use the same concentrations for both condition, then you could determine which of those might be effective or toxic for the cells.
Ideally you could also have a control where you treat your cells with the transfectant but not the oligonucleotides.
Best
Gilles
Dear Mohammad,
It is important to perform the miR negative control transfection at all the mentioned concentrations. Moreover, I suggest you to clone any psudo gene (scrambled sequence) in the same plasmid which your are using for your desired gene expression and to use it as a negative control.
Hi Mohammed,
You need a negative control miRNA mimic of identical chemistry. This will test the non-specific effect of the chemistry. A decreased expression of your target could be due to toxicity of mimic chemistry and not miRNA sequence-dependent. Ideally, you should normalize your target mRNA levels in the 10nM, 20nM and 30nM conditions to miR-145 and the negative control mimic respectively to show that the mRNA levels decrease in a miR-145-dependent manner. This is how I normalized my data in my miR-181 study: http://www.ncbi.nlm.nih.gov/pubmed/23524334
The best negative control would be a scrambled miR-145 sequence.
Hope this helps.
Jean-Charles
you can co-transfect miRNA mimics with reporter, using Lipofectamine 2000 or 3000. I'd recommend to do 3 concentrations as you planned, typical range 5-30 nM. for neg control- use at least one neg control sequence, ideally at the same 3 concentrations, plus untransfected cells
HELLO
For in-vitro experiments, negative control is important and the concentrations for negative control and mimics should be same for determining its toxic effect for the cells.
regards,
Shilpi
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