I am trying to figure out the expression profile of a bacterial gene in different condition using real time PCR. I have two doubts reagarding the two of my observations as i am handling the real time system for the first time. Firstly i am getting a conc. of primers (For. and Back.) - 10 nM each in standardization, which I guess is slightly lower of what one suppose to get in these analysis. On increasing the concenteration even to 25 nM the self amplification and dissociation attributes do not remain consistent. Though they are very much consistent in 10 nM conc. of Fw and Rv.
Secondly, I am not able to get significant amplification of the cDNA from my samples with 10 nM conc. of primers. It is happening in both the cases, as when i put 5 microgram/50 microlotre RNA for cDNA conversion as well as 20 microgm/50 microL (which is 4 times higher than recommended). I doubt that the cDNA might contain too less of my target transcript or its time to order new sets of primers.
No significant amplification in real time