Hi
can someone help me analyse my data
i have performed real time pcr for 2 sets on day 1 Average ct values are given
target gene
control 22.9,,,,, 24.0
treatment 24.6,,,,, 24.9
Gapdh
control 17.5,,,,,, 18.3
treatment 18.2,,,,, 18.9
when i did again real time pcr on 2 different sets i obtained different ct values though pattern is same.average ct values given
target gene
control 21.2,,,,,,, 20.9
treatment 24.9,,,,, 23.3
gapdh
control 16.4,,,,,,, 15.5
treatment 17.8,,,,,, 17.2
How do i analyse both of them together because if i club all the 4 exp together SD is quite high due to difference in their CT values. DO i use thermo method (separately do delta CT for each exp and in end take average and SD) but does the data normally distributed.
Help will be highly appreciated.
Thanks a lot
It is difficult to interpret the data with only the 'extremes' of your Ct-values. However, It looks like the effect size of your experiments are really different, and simply recalculating the data will not solve the problem; delta-delta Ct for your first experiment is probably ~0.7 (which corresponds to a 1.6 fold change; Ct value goes up, so this is a down regulation) whereas the delta-delta Ct of your second experiment ~1.5 (which corresponds to 2.8 fold change down). (I calculated it by using the middle of your extremes.)
thanks for the reply.
can i take this as an increase though the levels vary between 2 exp set....
actually my treatment is viral infection and it vary between exp and percentage infection is also low....
can i do Delta ct taget - gene ...or which way you did it
I estimated the expression by the delta-delta Ct approach. You can find more info in: Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-408
Please note that you have a decreased expression, because your Ct values go up!!
Thanks once again ...ya it goes down...my mistake...will surely go through the paper
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