So Can I use 5X HOT FIREPOL PROBE ROX for miRNA detection? I´m get used to use that FIREPOL for mRNA detection, but i never tried that for miRNA...THX
The main difference between mRNA and miRNA is in the reverse transcription since in the TaqMan system every miRNA requires a unique stem-loop primer. To the best of my knowledge the real-time PCR can be the same as it follows the amplification-dependent release of FAM fluorescence from its quencher relative to the constant reference ROX dye in the reaction. There is a mild dependency on amplicon length, but TaqMan probes for both mRNAs and miRNAs are supposed to generate similar amplicon lengths.
As a side note, if you use a method based on artificial polyadenylation followed by universal-tag-oligo-dT-driven reverse transcription, you can use the much much much cheaper option of SYBR Green in combination with plain oligos for both miRNAs and mRNAs from the same RT - no worrying about different RT efficiencies! If you are only looking at a few genes, go with TaqMan, but if you are planning more than, say, 10 types of probes, the cost gradient can be about 100-fold! The downside is that you have to include more controls. Which is also the upside :-) Let me know if you want a protocol.
Good luck!
Thank you so much.
So do you think that there is no difference between using the FIREPol or TaqMan universal MasterMix for miRNA PCR?
I´m plannig to detect only 2 types of miRNA, so I think that I´m going to use TaqMan technologies.
I really appreciate your help.
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