qPCR Exp. Design - Reruning Exp. Control Samples - (Nov/18/2007 )
Hi Everyone,
We are running a study to monitor the expression of a certain gene in the rodent brain induced by various training procedures. Here is the setup:
Training Type 1 (n=6) (6 experimental replicates)
Training Type 2 (n = 6)
Training Type 3 (n = 6)
Controls (Home Cage) (n=6)
We have one gene of interest and one control gene (GAPDH, no changes in Ct induced by training). We run each reaction in triplicate (so each experimental replicate (n=6) is run in triplicate for our gene of interest and in triplicate for GAPDH. We calculate relative expression using the standard Delta Delta Ct method. The only differences is that we use the highest delta Ct from our control samples to subtract from each delta CT from all the samples.
When I run the samples I always run the controls. So for example if I run the PCR on Training Type 1 and Controls one week, the next week I'll run Training Type 2 and the Controls again. What I am wondering is if it is necessary to rerun the controls every time I run a new training set or if I could just reuse the old control values. Thanks.
In Real-time PCR, absolutely you should use the control gene along with your target gene in every experiment. Otherwise the results won't be accurate.
We are running a study to monitor the expression of a certain gene in the rodent brain induced by various training procedures. Here is the setup:
Training Type 1 (n=6) (6 experimental replicates)
Training Type 2 (n = 6)
Training Type 3 (n = 6)
Controls (Home Cage) (n=6)
We have one gene of interest and one control gene (GAPDH, no changes in Ct induced by training). We run each reaction in triplicate (so each experimental replicate (n=6) is run in triplicate for our gene of interest and in triplicate for GAPDH. We calculate relative expression using the standard Delta Delta Ct method. The only differences is that we use the highest delta Ct from our control samples to subtract from each delta CT from all the samples.
When I run the samples I always run the controls. So for example if I run the PCR on Training Type 1 and Controls one week, the next week I'll run Training Type 2 and the Controls again. What I am wondering is if it is necessary to rerun the controls every time I run a new training set or if I could just reuse the old control values. Thanks.
Right, I understand that it is necessary to run GAPDH everytime I run a new sample set, but maybe I wasn't clear enough in my earlier post. What I am wondering is whether it is necessary to run my experimental controls (no training) animals every time or if I can use the old values. Thanks.
Yes. Still it is necessary to include your control in every set of experiment. Because. gene expression for your target gene as well as control gene in your experimental control (calibrator) may have some variation in different time points. Also, we don't know the efficiency of RT reaction. In one week, it may be 90% efficient (i.e. 90% of RNA were converted to cDNA); an other week it may be 80% efficient. Those differences will be nullified when we use experimental control every time.