Hello, I'm working on detecting miRNAs using hydrogel microparticles with probes incorporated in it.
Recently, I found an article about the relation between melting temperature and specificiy of miRNA assay.
It is told that as assay temperature goes down, specificity of miRNA decreases while sensitivity increases.
I want to know clear mechanism of this phnomenon.
Thank you!
The mechanism of your detection is that your probe has to be comlementally annealed onto your detected miRNA , RNA or DNA. This annealing is determined by melting temperature. At an assay temperature lower than melting temperature, your probe much easier to not only anneal to your target mIRNA but also to the other RNA-the result of increase sensitivity. Since your probe should be 100% match your detected miRNA and have the highest annealing temperature in comparison to that of other non-specific one, as your analysis temperature increase, your probe lose the annealing to the less matched RNA and finally only your target miRNA can be annealed with- a result of increase of specificity. Of course if the temperature higher that your melting temperature, nothing can be detected with your probe.
Thank you for answering!
Is melting temperature as you mentioned above is same meaning as assay temperature??
Melting temperature is calculated based on your probe sequence, it varies depend on the buffer conditions and calculation methods. assay temperature is the temperature you used in your assay.
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