I am working with miRNA expression in canine cancer cell lines. This particular question refers to my reporter assays to test the miRNA and 3'-UTR target sequence binding in cancer cells. I am co-transfecting cells with reporter vectors expressing 3'-UTR and related miRNA precursor cloning vector in 6-well plate. I need to optimize the concentration of these vectors. Do anyone have an experience with relevant assay so that I can get some suggestions about the optimum concentrations of these vectors for co-transfection. Thanks.
You don't mention what your reporter is, luciferase? GFP? Alk phosp? The minimum amount of reporter vector depends on the sensibility of your assay, for GFP assays the minmium is dictaded by what is visible but for a LUC reporter the dinamic range is wider and the minimum is determined by the noise from the empty vector control and the strength of your promoter/enhancer in the particular cell line you're transfecting. From this, minimum level of detection you can try to titrate in 1-fold and upwards (molar) of your silencing miRNA vector. From whatever results you get from this initial run, you could scale up both plasmids to a more robust signal and for the miRNA you can narrow down to the more interesting concentrations or higher. If you can get your hands on a set of reporter/miRNA pairs that are known to work, then it would a nice parallel control to have. - Just some thoughts.
Thanks for your answer. One of my vectors contain luciferase and another has GFP and they need to be co-transfected.
Hi Farruk, I agree with Oscar regarding the usage of known target 3`-UTR reporter as a positive control for optimization. I just want to add something about the miRNA precursor vector. Sometimes miRNA pri-/pre- vectors take longer time to express mature miRNA in cells where you are transfecting. If you get enough miRNA expression within 24-48 hours, it should be OK. If not, and your 3`-UTR reporter (luciferase) vector contains stronger promoter (e.g, CMV) than miRNA precursor vector, you might not observe any detectable effect of miRNA at certain time point since your 3`-UTR reporter vector will have enough expression before the initiation of miRNA expression. I had that kind of problem earlier. I constructed doxycycline-inducible miRNA expression vector (containing pri-miRNA) that effectively repressed target gene expression, but I could not use it for 3`-UTR reporter assay because of this problem. In such cases, its better to use miRNA mimic for reporter assay and cells with high transfection efficiency (293T). Also, endogenous miRNAs might dilute the effect of pre-miRNA by competing with 3`-UTR reporter.
Hello Ruhul,
I have also an aim that matches your last sentence. Since I have evaluated the comprehensive expression profile of all highly characterized miRNAs in my cancer cell lines, I want to check the endogenous miRNA effects as well. I also agree with the relative strength with different promoters used in constructing vectors. The construct with 3'-UTR clone has CMV and SV40 enhancer for dual reporters while the precur- miRNA vector has H1 promoter. This is why I may need to optimize the concentrations of these vectors when co-transfecting cells.
You know, miRNAs are usually transcribed from Pol II promoter. So, using the pol II promoter containing miRNA vector (like CMV) can represent endogenous Pre-miRNA processing into mature form. It was found that, incorporating the same mature miRNA sequence into pol III vectors exhibited decreased activity than pol II vector. Since, your precursor vector contains pol III promoter (H1), you can try different ratio like 1:2, 1:5, 1:7, 1:10 etc and choose the best one. Its just a thought but you can play with molar ratio this way.
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