I am measuring miRNA levels with qPCR using taqman probe (regular, no detailed information on it). Recently I introduced some miRNA with GFP-tag to my system, and did qPCR with the taqman system.
My concern is: are there any fluorescent overlaps between GFP and taqman probe?
What fluorophore are you using for taqman probe?
I think one of the most common fluorophores for the Taqman probe is FAM with an emission wavelength 520 nm and TET with 536 nm. They both are quite "green". GFP emission peak is at 509 nm, if I am correct.
So there could be a fluorescence overlap.
Are you doing qPCR or RT-PCR? For measuring miRNA levels you would need reverse transcription and I assume you use the stem-loop protocol?
If you purify your RNA correct for RT-PCR there should be pretty much no protein (GFP tag) left in your sample to begin with. Do you want to measure your tagged miRNA with the TaqMan assay? If yes ... how do you intend to purify your sample?
Taq amn probes can be ordered with something other that FAM, like TET- 521. 536 (excitaion-emisson), JOE-520. 548 or VIC. ~555(emission). What machine are you using, atleast 3 filters should do your work. Generally ROX or TAMRA are used for internal control so your probe-primer should have a different label that these two.
Dr. Ulrike, even if GFP is tagged and its a fusion construct, then GFP along with the miRNA will be transcribed and should be a single fusion miRNA, why should it be in the protein? If its not an IRES GFP where GFP will be expressed separate as opposed to a fusion construct, is there a problem of getting a fusion RNA?
So any miRNA which is made will have a GFP tag along with the sequence of the gene/miRNA of interest.
Please correct me if I am missing something.
http://www.ncbi.nlm.nih.gov/pubmed/20554852 I am not sure if the link is very helpful.
Kalpita if I did not misunderstand this he adds miRNA which come pre-synthesized with GFP as the tag. There is no such thing as transcribing miRNA with a protein translated to it in a cell. The miRNA processing is mRNA based, there is nothing attached to it anymore afterwards that could be translated into GFP.
Dr. Ulrike, thanks a lot for the concept. I surely was missing things here.
Quan, I am not sure how much helpful this link mught be but you can have a look.
http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1000996
Quan what you can do is something usually done in CHIP-Chip, you can basically immunoprecipitate your GFP with an antibody linked to a magnetic bead (Dynabeads for example), this binds to a magnetic column. The result is usually treated with trizol or Stat60 to get rid of the bead and the protein but what is left is pretty much exclusively what your labelled miRNA was.
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