Primer concentration and standard curve slope in "Relative Standard Curve M - (Apr/28/2007 )
I have two concerns about the "Relative Standard Curve Method":
1, Is it necessary to use the same concentration of primers for the gene of interest and the house-keeping gene on the same plate?
2, Is it necessary to optimize the conditions so that the slopes of the standard curves of both the gene of interest and the house-keeping gene are close to each other? If so, how close is satisfactory?
I'll appreciate any comment.
Dadi
-dadiame-
QUOTE (dadiame @ Apr 28 2007, 04:29 PM)
I have two concerns about the "Relative Standard Curve Method":
1, Is it necessary to use the same concentration of primers for the gene of interest and the house-keeping gene on the same plate?
2, Is it necessary to optimize the conditions so that the slopes of the standard curves of both the gene of interest and the house-keeping gene are close to each other? If so, how close is satisfactory?
I'll appreciate any comment.
Dadi
1, Is it necessary to use the same concentration of primers for the gene of interest and the house-keeping gene on the same plate?
2, Is it necessary to optimize the conditions so that the slopes of the standard curves of both the gene of interest and the house-keeping gene are close to each other? If so, how close is satisfactory?
I'll appreciate any comment.
Dadi
with concern of relative quantification are.....
1. primer concentration may not be the same but make sure your PCR efficiency of gene of interest and house keeping gene are above 95% and they are the same (as close as possible).
2. once the conditions fixed, use that condition consistently through out your entire experiment.
-Hadrian-