Detecting miRNA mimics by RT-PCR? - (May/02/2013 )
I will inject miRNA mimics in mice.
I would like to detect wether the mimic gets to the target organ and what is the best dose and the best injection route to use.
Is it possible to detect the miRNA mimic with an RT-PCR even if the mimic is a double strand RNA?
Anyone tried it before?
If it is not possible, anyone has a better idea how to do it?
You can detect miRNA in tissues, so it should be a similar process to detect a mimic -- unless the altered chemistry of the mimic interferes with the assay. It might be dificult to distinguish a mimic from the background of natural miRNA. If you are using a PCR method, the high-temperature steps should melt the miRNA duplex apart.
For a fluoresence in-situ hybridization assay, you might try something like this:
Lagendijk AK, Moulton JD, Bakkers J. Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues. Biology Open 2012 BIO2012810; Advance Online Article April 27, 2012, doi:10.1242/bio.2012810
Thanks for your answer.
I don't know if the chemistry of the mimic will interfere with the assay. What I was told is that the problem might be the fact that de mimic is a double strand and that the reverse transcription is usually done on single strand RNA.
I might just test it in transfected cells before going all out on animals!
How about heating your cell lysates just before RT-PCR? That should blow the duplexes apart. You might need to flash-chill to near RT.
Great idea, if it doesn't work the regular way, i'll try this!