increasing sensitivity and reducing primer dimmer - (Oct/31/2006 )
Hello everyone:
I´m performing real time RT-PCR with SYBR GREEN but I need to increase sensitivity because my primer pair doesn't detect some positive samples from field, but they work really well with positive controls. Also they are good because they don't form primer dimmer. I have been using other pair with more sensitivity but they yield lots of primer dimmer. What can I do to increase the sensitivity of one pair or to reduce the primer dimmer formation of the other one?
Thanks!!
do they have equal efficiency?
also....as to how to get the best results and choose a primer pair: have you titrated them each to determine optimal amplification? you may need to titrate the amounts of primer you add in order to find the best combination, and then calculate your efficiencies at that primer concentration
also....as to how to get the best results and choose a primer pair: have you titrated them each to determine optimal amplification? you may need to titrate the amounts of primer you add in order to find the best combination, and then calculate your efficiencies at that primer concentration
Hello!
I have titrated them and I did a matrix of different concentrations to find the best concentration of each primer, but they still do primer dimmer. I use them at 400nM (both) because is the concentration that has more efficiency
hmmmmm
I think I would find a new primer-pair. you need to get rid of the dimers, and it sounds like you've tried all the good tricks. dimers will interfere with your results, though
For your "positive controls" are you using an endogenous template as your control eg. GAPDH? Otherwise you may have some problems with the purity of your RNA such as contaminating RNase or PCR inhibitors.
Hello:
I don´t use an endogenous control because I don't know how to do it and I think is easier to design new primers than starting to use an endogenous control. When I use the primers with my positive controls I don't get primer dimmer.
Thanks for your replies
I'm confused 
how can you be doing realtime without an endogenous control? how do you determine your relative values? what controls do you use, then? even if you run a standard curve every time, you still need an endogenous control, the same reason you need a loading control if you do a quantitative Western
how can you be doing realtime without an endogenous control? how do you determine your relative values? what controls do you use, then? even if you run a standard curve every time, you still need an endogenous control, the same reason you need a loading control if you do a quantitative Western
Hello:
I do Real Time with SYBR GREEN but I don't quantify. I only want to detect the virus. When I will have a good pair of primers I will do dilutions of the same positive sample and from there, I will do my standard curve to quantify ( in relative values). It's really difficult for me to quantify in absolut values the numer of copies I have because I don't know how many virus I have on my positive sample. In the future I want to infect a cell culture with a known virus concentration and construct a better standard curve.
The positive samples ( controls) I am using are from a laboratory and they have purified virus. Also I use positive samples from the field, wich have less quantity of virus and that's when I have problems. As the field samples have less virus, my primer pair is not so sensitive and doesn't detect the positive samples, only my controls( from the lab). That's because when I choose this pair, was based on the results I obtained with my positve controls from the lab ( purified virus)
Based on the things you told me and that I think I can't imrpove the sensitivity of this primer pair, I decided to design a new pair.
I think you should consider running a standard curve with plamid DNA as your target to make sure you can assess the ssensitivity of your reaction. The other thing to worry about is PCR inhibitiors in your "unknown" samples. Thus, the problem may not be with your primers, but with your method of NA purification.
Hello:
I understand what you say but I already know that my assay has sensitivity, what I want is to increase that sensitivity, wich I don´t know if it is possible or if it's better for me and less time consuming to change my pair of primers and design a new one. The method I use for RNA extraction is a silica column method and, I also used to compare, the TRIPURE method. With both methods I obtained the same sensitivity. About the PCR inhibitors, I will consider this issue but I think is not probable because it was blood without EDTA or heparin