standard curve problem - standards concentration seems to be too low, but how? (Nov/29/2005 )
Hi there,
I have been performing my first RT-PCR runs. I am using LightCycler, sybrgreen and relative quantification with external standards. These are prepared by normal RT-PCR amplification of commercial cDNA samples which are afterwords gel purified. Then I measure DNA concentration with espectrophotometer and make dilutions just before the run.
As I started with the amplification of the housekeeping gene everything went fine, the standard curve was very nice. But when I tried with my target gene, the more diluted standards give amplification 0. The standards concentrations go from 10000000 to 1000 copies /ul. So I am having problems with 1000 and 10000. I have understood that this instrument should be able to detect this amount of copies.
Please, anyone who has had this problem, help!!! 
coco
Hi again,
I was thinking if the problem could be solved or at least alleviated by decreasing the annealing temperature, this would decrease specificity, and maybe allow amplification of smaller concentrations. I haven't had any primer dimers at all, not iven in the negative control (H2O instead of sample), so maybe this could help?
Does this make any sense?
What about changing some other parameters like Mg concentration?
Anyone can give an opinion?
coco
Hi Coco
As you told your Sandards 10^3 and 10^4 gives no result. I don't work with Light Chycler but I know it is unsing tree parameters from the curve to etamate the cp value. Your standards schoult reach the plato of amplification. You could increase the amound of cycles. What is the cp value of 10^5?
But you have still the cp of 10^5, 10^6 and 10^7. How do they behave? Is the slope OK. If the efficeienty is fine I wouldn't cange the program or MgCl concentration. And what about your samples are they within you Standard curve?
I have been performing my first RT-PCR runs. I am using LightCycler, sybrgreen and relative quantification with external standards. These are prepared by normal RT-PCR amplification of commercial cDNA samples which are afterwords gel purified. Then I measure DNA concentration with espectrophotometer and make dilutions just before the run.
As I started with the amplification of the housekeeping gene everything went fine, the standard curve was very nice. But when I tried with my target gene, the more diluted standards give amplification 0. The standards concentrations go from 10000000 to 1000 copies /ul. So I am having problems with 1000 and 10000. I have understood that this instrument should be able to detect this amount of copies.
regards
Montgomery
Please, anyone who has had this problem, help!!!
coco