I am having some issues with primer optimization for qPCR. Typically, I first test various concentrations of primers to figure out which concentrations gives the best melting curves and the lowest Ct values. Then, I perform a standard curve using 10-fold dilutions of my template (at least 5 dilutions) to determine efficiency. Recently, all my standard curves plateau in the last two dilutions (only the first three dilutions give a linear curve). I also tested primers that have previously been optimized in our lab and I get the same curve (with plateau). So, I'm thinking it must be something I am doing wrong. Not sure how to approach this. Would appreciate any recommendations.
Usually I design several pairs of primer for each gene that I'm interested in and run the primer optimization at once, then pick the best one for the qRT-PCR (single peak in the melting curve, efficiency 90-110%, Tm close to Tm of the internal control primers,...). If you see the amplification curves flatted out in the last 2 dilutions, you might not have enough template in the reaction. I'd recommend you to try 4-fold dilution instead of 10-fold dilution. This will keep your reaction in the detectable range.
Usually I design several pairs of primer for each gene that I'm interested in and run the primer optimization at once, then pick the best one for the qRT-PCR (single peak in the melting curve, efficiency 90-110%, Tm close to Tm of the internal control primers,...). If you see the amplification curves flatted out in the last 2 dilutions, you might not have enough template in the reaction. I'd recommend you to try 4-fold dilution instead of 10-fold dilution. This will keep your reaction in the detectable range.
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