Hi all - I am testing some probe/primers assays to see if I can multiplex one of my ref gene (PPIA) with a target gene '(Tas1R2).
This is a typical amplification curve obtained - I have diluted the ref gene assay 1:2 to reduce possible inhibitory competition with target gene.
Still the target gene multiplex amplification curve show a shoulder that I do not see in the singleplex amplification curve. Any idea of what causes this ? Dimers between assays ?
Many thanks for your suggestions !
Look at the scale on the Y axis. You have basically 0 expression and seeing a lot of "noise" around 0.
Madam this is not a direct answer but its a prespective on reactions. normally chemical reactions there is a linear curve. reactions in colometry or chemistry labs.
but biological reactions they have a sigma shaped curve, eg enzymatic reactions also the way oxygen combines with Hemoglobin, after one molecule of oxygen binds to hemoglobin this hemoglobin with one molecule of oxygen has a higher affinity for the next incoming oxygen. here also the curve is sigmoid shaped.
Am not qualified to comment more than this.
Katie A S Burnette - thanks for you reply. My Y axis is for delta Rn , that is fluorescence value, and not "expression" value... A sample with high amount of target could give low increases in fluorescence if the reaction is inhibited, if there is high reference dye level etc...
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