interpretation of results of relative qRTPCR - please help (Jul/30/2008 )
hi all
I have been doing Sybr Green real time . I use the deltadeltaCt method to calculate differences between my treatment samples and control after normalizing with a housekeeping gene. My problem is in interpretation of results.
when i use the excel for my control and the target gene i take two values for the 2^-ΔΔCt factor.
for my control is 1,021012126
and for my target is 0,615572207
that means that if control is 100% then target is ~60%???
does this mean that my target gene is downregulated about ~40%???
thanks
tasos
Yes, 2^-deltadeltaCt gives you RQ which is the relative quantity of the target sequence present.
I have been doing Sybr Green real time . I use the deltadeltaCt method to calculate differences between my treatment samples and control after normalizing with a housekeeping gene. My problem is in interpretation of results.
when i use the excel for my control and the target gene i take two values for the 2^-ΔΔCt factor.
for my control is 1,021012126
and for my target is 0,615572207
that means that if control is 100% then target is ~60%???
does this mean that my target gene is downregulated about ~40%???
thanks
tasos
Actually it does not mean anything. You can not say anything remotely surely when comparing with only one housekeeping gene. It could mean that this particular housekeeping gene is upregulated under your circumstances, or that your target gene is partly downregualted and the chosen housekeeping gene is partly upregualted.
You should use 3 housekeeping genes.
thank you both for your reply!
let me be more specific: I have 2 samples. 1 from a healthy control and 1 from a patient. I manipulate them and then ive got 1 cDNA from healthy control and 1 from patient, prepared from the same amount of RNA.
then i do real time pcr (every reaction run triplicate) and i also use some dilutions of cDNA.
I use GAPDH in healthy control and GAPDH in patient as control. (2 reactions).
and my target gene in healthy control and target gene in patient. (another 2 reactios).
when i put my Ct's on the excel, i get for the 2^DDct factor the numbers i mentioned before.
I know that the result may not be statistically significant or biologically significant (yet)
BUT do i interpret it correct when I say that my target gene in patient is 40% downregulated in comparison with target gene in healthy control?
YES, that is the correct interpretation. More housekeeping genes would be good, of course, but then, if you don't see a change in the one housekeeping gene you use now between the experimental conditions, and you got a good method for RNA quantification from the beginning, at least I think you could wait with using 3 HKG:s. Good luck.
Did you check the similarity of Amplification Efficiency between your target and reference?