Try to describe your protocol? Are you using the one from trizol or have you changed it?

I would try Immunoprecipitate in native conditions with GFP antibodies and then perform the qRTPCR from the precipitated.

Several protocols are available on line.

Good luck

I also recommend you to immunoprecipitate your sample using an antibody against the protein of your complex and then purify miRNA/RNA and perform qPCR

Carlos, could you add the links to those protocols?

Las: I followed the regular procol for Trizol, didn't do any modification.

Carlos, Lidia:

Immunoprecipitation could able to isolate pure GFP-miRNA fusion species.

The problem is how do I process the downstream procedure to isolate miRNA or remove GFP? or I do not need separate miRNA from fusion protein? If though, does GFP influence the result of qPCR in tep of polymerase chain reaction or fluorescense detection?

There are some kits for immunoprecipitating RNA-protein complexes (for example: Magna RIP, Millipore). Some steps are included to separate the complex and even the protocol add steps for downstream processing of the samples, including qPCR. In a general protocol, the addition of Trizol to the immunoprecipitated samples would cause that separation.

Lidia, mine is fusion protein, I used vector with zGreen and ligated miRNA into downstream of zGreen. Transfected construct into HEK293T, and trying to account the miRNA levels with RT-PCR. But, eventhough my cells were glowing with green, there's no miRNA elevation in my overexpression system. So, I am trying to fix the problem from RNA purification step. Is proteinase K helpful to digest GFP from fusion-complex without any influences on miRNA?

You can use RNAGEM tissue plus RNA extraction kit (ZyGEM, Hamilton, New Zealand).

It basically degrades all the protein and DNA leaving only RNA molecules in the extract. May be helpfull for you

I've never used Proteinase K for fusion proteins like yours, but I'm not sure that the RNAGEM tissue plus RNA extraction kit would purify miRNAs or other small RNAs...

Keene, Komisarow, & Friedersdorf, Nature Protocols, 2006 is a nice protocol for protein IP followed by RNA extraction. You need to make sure you have enough starting material though. Also, if you want to try just extracting miRNAs again (without IP) I like the quiagen miRNeasy micro kit.

RNAIP should work, for that you have to use special beads, as for biotinylated miRNA people use streptavidin

I also recommend IP followed by miRNA extraction with miRNeasy mini kit (Qiagen)

"Lidia, mine is fusion protein, I used vector with zGreen and ligated miRNA into downstream of zGreen. Transfected construct into HEK293T, and trying to account the miRNA levels with RT-PCR. But, eventhough my cells were glowing with green, there's no miRNA elevation in my overexpression system. So, I am trying to fix the problem from RNA purification step. Is proteinase K helpful to digest GFP from fusion-complex without any influences on miRNA?"

Hi Quan, am I correct in understanding that you have cloned a pre-miR sequence downstream of the GFP gene? If so, there would not be a fusion protein as the GFP part will be translated separately and the pre-miR hairpin should be processed by Dicer (either during or after transcription). (Something similar was done by this group http://www.retrovirology.com/content/5/1/117 , although in this case, the pre-miR- hairpin was upstream of the GFP gene).

If my assumption is wrong, and you do indeed have a GFP-miRNA fusion protein, Proteinase K digestion might help to remove the GFP part.

Hope this helps!

Srinivasa, I think you are correct. GFP protein and mature miRNA should be chopped by Dicer during RNA processing.

But I still don't understand that In my system, GFP expression levels (Fluorescence) are not correlate to miRNA levels (qPCR).

Where, when did I lose my RNAs?

Maybe if you use denaturing conditions for the IP the GFP will not be fluorescent any more, and you would not need to get rid off it.