hi

for transient ko antimir http://bioinfo.appliedbiosystems.com/genome-database/mirna.html they have most of them

then I used RNAimax or lipofectamine 2000 (less efficient) on mESc and hESC and it work great for upregulation, downregulation was ok 0.3 to 0.5 fold to ctl condition (applied has also taqman qRTspecific mir assay).

for stable ko design shRNA against the premir and use integrative vector or lentivirus (must have antibiotic selection k7) but check literature for more.

Well this is the simpliest way I know, btw antagomir are cholesterol combined for in-vivo administration

enjoy your mir research goodluck

look up "miRNA sponge" for transient or permanent miRNA KD

Dear Nasim,

I believe any of the tools suggested above can help you! But if as my personal advise, I would recommend microRNA Hairpin Inhibitors.

And Dharmafect or Lipofectamine 2000 both can work well for transfection. Provided you must check its an Easy to transfect cell line or not? In that case Dharmafect might not work but it usually keeps high cell longivity and low toxicity.

Good Luck!

Gaurab Banerjee

Continuation to Nasim's question, does the method suggested here good for neurones too?

Dear King Hwa Ling,

This is a good question there is a product from Dharmacon Accell which is capable to show good transfection in Neuronal cells as siRNA. The same protocol works well for the miRNA mimimcs as well as hairpin inhibitors. There shall be a delivery media you will receive with the product.

For siRNA an ex-vivo experiment was performed.

http://www.dharmacon.com/uploadedFiles/Home/Support_Center/Application_Notes/accell-brainslice-app-note.pdf

Initially this technique was applied for siRNA and found good success. Later it can now be available for miRNA also.

http://www.dharmacon.com/product/productlandingtemplate.aspx?id=2335

Hope it works for you!

With best wishes.

Gaurab

Thanks for your suggestions

I hv the same related question, after transfection with miR inhibitor, can we check the inhibition by real time PCR or any other method?

Yes Surely you can. There are Solaris qPCR sets for each individual gene. It is a complete set of primers and qPCR reagents that will be required for a specific known gene regulated by your miRNA of interest.

Good Day,

Gaurab

I did by qPCR, but didn't find any difference after transfection? I used Qiagen miR inhibitor and transfection reagent...

I believe then you must observe the response at protein level. With and without controls and also timeline is important.

There should be a good upregulation of the gene if the correct miRNA was adequate in the sample.

Cheers

Gaurab

Western Blot or Flow Cytometric analysis.

Thanks Gaurab

Dear Nasim,

I would suggest you to use miRNA-specific LNA exiqons. They are very specific and you need to transfect a very low quantity to get inhibition. Please let me know if you would like me to add something to that.

Regards

Sankar