In qPCR optimization I have a gene that has low expression and my samples dilution (1:3) gives me a calibration curve with low R^2. And I have another gene that gives me a nonspecific band amplification. Nevertheless I already optimized other genes for the same conditions as the reference gene (inicial conditions). So can I increase primer concentration for the fist gene and lower primer concentration for the second gene? keeping the initial conditions for the reference gene?
Yes, you can use different primer concentrations for the target gene and reference gene in PCR optimization. However, the concentrations are within an optimal range for each primer set to maintain specificity, efficiency, and balance in amplification. Experimentation and validation are crucial to achieve reliable and reproducible results.
In proncipal Yes, Because reference gene usually have high expression. For instant, β-actin (as reference) in teleost have very high expression level in all tissues particularly in gonads tissue. In this case you can use different primer concentrations for your target gene and reference gene. However I suggest to use same concentration if possible for avoiding further explanation issues.
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