Due to a low miRNA expression, I would like to preamplify our samples. I currently have taqman reagents, and taqman miRNA primers. Could I use the qaigen MiScript PreAmp PCR kit for this using taqman small RNA Primers?
hope this link is useful
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753550/
regards
hope this link is useful
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753550/
regards
I know pre-amplification approaches exist, but I would tend to caution against using them unless absolutely necessary: the amplification methods are usually linear (or mostly linear), because this is important, but they're still amplification methods. In other words, if you can amplify your target via a pre-amp kit, you could probably amplify it equally well (albeit perhaps noisily) via regular qPCR. And you can account for noise by simply increasing the number of replicate wells.
It is rare to find a target that cannot be detected by PCR, but that CAN nevertheless be amplified to (subsequently) detectable levels.
Secondly: if your miRs are present at such low levels, how biologically-relevant do you think they truly are? A two or three-fold increase in expression of a miR that is basically not really expressed at all is something that could well just be stochastic noise, whereas if your expression increase is orders of magnitude greater, then the basal level of expression doesn't really need to be measured that accurately, it could just be presented at 'several thousand times lower'.
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- Biological Science
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- Nucleic Acids
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- microRNA