New to qPCR - (Jul/28/2008 )
I'm new to qPCR and need some advice on optimization of my reactions. I will be using SYBR Green chemistry with delta delta Ct relative quantification. My template is bacterial RNA. My endogenous will be 16s and I have 7 other primer sets to look at gene expression in various in vitro conditions.
I've read that I need to do primer optimization for each primer set, and that's fine. I've also read that I need to optimize my template concentration. So, do I need to do each primer concentration with each template concentration, or do I just do primer optimization FIRST and then to template optimization?
The second part of that is that I'm going to be doing multiple treatments for growth conditions, so do I need to then optimize all of the above for each growth condition, or optimize for primers and template and then use those same concentrations for each growth treatment? Having to optimize for each growth condition seems like entirely too much to have to do, but I'm just not sure.
I hope all that made sense.
Thanks for any information.
you don't need to optimize primer and template concentration for each growth condition. you should just optimize the condition with a few samples and then use it in all the entire assays. drawing an standard curve usually helps in assessing the efficiency of the assay.
1. optimize your primer concentrations and annealing temperatures. preferably should be about the same as your endogenous gene.
2. just use the template you have now to do serial dilutions. pick the best range for your experimental samples.
3. nope don't optimize for all conditions. your results will be skewed then. use a normal sample/untreated control RNA for your optimizations.