Hello everyone!
As I had a bad experience with pre-mir transfection in Luciferase Assays, I would like to buy directly mature mirna mimics (and antagomirs), which look like to be in general more efficient.
Did anyone have nice experience with these? More precisly, I would like an advice on the BRAND of the molecules.
I would like also to know at which concentration you used and after how many hours from the transfection you should collect the cells. Bibliography on this topic is large and contradictory, so, I am looking for someone that could simply tell me his/her experience!
I will do my experiments in HEK293 cells and I was planning to use the 3'UTR of HNF1B in the mirGLO (Promega) vector + mimics/antagomir.
Thank you!!!
I like Qiagen miScript miRNA Mimics, transfected with Lipofectamine RNAiMAX in optimem. Worked well for me. 50nm final concentration, allowing 48h for knockdown. Alternatively, miRIDIAN microRNA Mimics from Dharmacon are also good.
Hello Pierbruno
I have worked in primary cell that are a little difficult to transfection that cell line. I use miScript miRNA mimics and it works very well. For transfection, I use Hiperfec transfection reagent both from Qiagen with a concentration of 15nm.
My experience is in culture plate-6 wells. First, I do the mix transfection according to manufacturer instructions and add approximately 150 ul on well culture for all cell surface. After, I full with culture media untill 600 ul. Incubation for 6 hours. Then, full untill 1ml of culture media for 18 hours more, allowing 48 hours for knockdown.
Regards
Hello,
I use Dharmacon mimics and inhibitors. I use 0.5 to 2 ul of 20micromolar miRNA mimic or inhibitor of miRNAs. I also used the transfection reagent from the same company. You can find information about this transfection reagent here:
http://dharmacon.gelifesciences.com/transfection/dharmafect-1-transfection-reagent/
For co-transfection, the protocol is so easy. You can add miRNA mimic or inhibitor as well as your reporter to the above-mentioned reagents. You can adjust the concentrations based on the DharmaFECT 1 Transfection Reagent instruction.
HEK-293 cells are also very easily transfectable. Therefore, after 48 h of transfection, you can harvest the cells. Take note that, since these cells growing so fast, it is better to start with 40% confluence.
mirVana mimics and inhibitors. deliver with RNAiMax
https://www.thermofisher.com/order/catalog/product/4464065?SID=srch-srp-4464065
use naturemiRI microRNA inhibitors they work the best. They are plasmid-based microRNA inhibitors and you do not need to flood the cell with toxic oligos. The plasmid is easy to transfect cells using your lab method and cell retain the plasmid for several passages. The results are very specific. With all microRNA inhbiitors you must screen for off-target effects and the products from naturemiRI are proven to have no off target effects.
https://naturemiri.com/learn-more/
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