You need a well defined standard of your target, as mentioned above already. Using a serial dilution you can test for the analytical sensitivity of the actual PCR protocol. If you have a cloned standard at hand, than try to calibrate to concentrations being equal to 1 copy of your targt per reaction in the highest dilution. Do not test sensitivity in a single run, only. Rather, perform a number of replicates. With the highest dilutions 10 replicates on two or three Independent PCR-runs could be helpful (especially when working with high dilutions/low target concentrations, stochastic effects start to influence sensitivity). Of note, you should define, how sensitive your PCR needs to be. Otherwise you may end up in hunting for a sensitivity which is acutally not required (i.e. it is not always required to detect a single copy per reaction).
Many Parameter affect PCR sensitivity like concentration of Primer, Enzyme, dNTPs, MgCl2 etc. You need to check all that parameter step by step with know no of organism in sample (Spiking Exp.). Once you done that then sensitivity of PCR, more emphasis should be put on testing of several primer pairs than on the extensive screening of reaction parameters.
By trial and error
i would make serial dilutions of the template and see at what point you can no longer detect a product
i would reduce the number of cycles to see when you can no longer detect a product
of course the method of detection will have an influence on how sensitive a particular reaction is , qpcr or radionucleotide incorporation are going to me much more sensitive than a standard ethidium bromide stained gel
You need a well defined standard of your target, as mentioned above already. Using a serial dilution you can test for the analytical sensitivity of the actual PCR protocol. If you have a cloned standard at hand, than try to calibrate to concentrations being equal to 1 copy of your targt per reaction in the highest dilution. Do not test sensitivity in a single run, only. Rather, perform a number of replicates. With the highest dilutions 10 replicates on two or three Independent PCR-runs could be helpful (especially when working with high dilutions/low target concentrations, stochastic effects start to influence sensitivity). Of note, you should define, how sensitive your PCR needs to be. Otherwise you may end up in hunting for a sensitivity which is acutally not required (i.e. it is not always required to detect a single copy per reaction).
Hi Manish,
By doing a dilution series of your (c)DNA as well as primer templates you can determine what the lowest detectable amount of your mRNA/DNA target as well as optimal primer concentrations. It is important to ensure there is a clean melt curve with the [primer] you choose and adjusting the melting temperature may also help ensure the best sensitivity. Best of luck.
Tyler
I agree with all the answer mentioned here in response to this question. Yes, we can say sensitivity is directly proportional to specificity. More sensitive means more specific to its experimental condition.
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