Help of Q-PCR - (Dec/12/2006 )
Hi every expert in the forum:
I am doing Q-PCR to access expression of the gene of interests. But a huge problem is facing me:
the primers (designed by Primer Express ABI) have created so strong background signal with SYBR GREEN I. To prove whether it is primer dimer, I performed Q-PCR using diluted primer with different concentrations 1/40 (I normally used for Q-PCR), 1/100, 1/200, 1/400/, 1/800, 1/1500, 1/2400. Also I did forward and reverse primers separately. the result showed that with the separated primers, no signal has been detected, but with the primer together, intense signal has come up around 25 cycle from concentration of 1/40 to 1/400.
I did my experiemnt with samples from reverse transcription and used GAPDH and PSA (primers which can access the quality of my samples) primer sets. They all worked fine. But with the primer of my interests only primer dimer has been generated (I checked this by running melting curve and running the product on the gel after PCR, which suggestted that this is primer dimer)
Then I ran the Q-PCR again using the optimized primer concentration 1/500. But no any signal been detected. I m sure that the primer won't creat dimer under that concentration and it seems no problem with the sample I made and I even re-ordered my primer just in case of contamination, but in the end, got the same problem.
Do u think the Primer Express softwarr is trustable. Did anyone came across the same problem with me. Could u give some recommendation to me
I would really appreciate u
I prefer primer3 is easier, fast, free and give really good primer set for choice. I know many people that have problems with ABI software.