Can someone explain me how it is possible that for some samples I have one positive and one negative replicate?
For example, during my last RT-PCR I loaded 8 ul/well of cDNA diluted 1:2 for MT1, and I used as housekeeping gene CDK4, loading 2ul/well of cDNA diluted 1:2. cDNA samples for the 2 genes were from the same mix. I have done 2 replicates for every gene.
The results of my RT-PCR are that CDK4 gives great replicates, and for some samples of MT1 I have one positive and one negativer replicate.
Moreover, the Ct of MT1 is quite high (after 34-35 cycles).
I cannot interprete my results, and eather find a solution to this strange problem...
Are you awared of a technique to "decrease" the Ct of a gene?