I run qPCR with, Control sample (target gene and HC gene) similarly with Treated sample?( target gene and HC Gene) and use livak methods for fold change, I got the value 0.17923 for 2^-ΔΔCt. my methods was like
Wild type APEC
ΔCt = Ct(csgA) – Ct(uida)
Silenced APEC
ΔCt = Ct (csgA) –Ct (uida)
ΔΔCt = ΔCt (Silenced) – ΔCt (Wild type)
Relative Expression rate =2–∆∆Ct
now how to interpret what is the fold change how to say its reduction or induction?
Hi Sayed,
Taken in consideration your results , you have a reduction of your target gene in comparison with your control genes. Using the livak method , when you have values between 0 and 1 , you usually have a reduction of expression . Because you have 0.17 , then 1/0,17=5.88 meaning that you should have a 5,88 fold reduction of your target gene under the experimental conditions
Good luck :)
Hi Sayed,
Taken in consideration your results , you have a reduction of your target gene in comparison with your control genes. Using the livak method , when you have values between 0 and 1 , you usually have a reduction of expression . Because you have 0.17 , then 1/0,17=5.88 meaning that you should have a 5,88 fold reduction of your target gene under the experimental conditions
Good luck :)
Dear Sayed,
Please be sure that your reference gene (HC) expression does not affected by experimental conditions. Usually it is additional experimental work before the main study. But if you haven't time and sourses for that work, please at least use 2-3 HC genes. Moreover it is very important to estimate reaction effectiveness for both target and reference genes to receive the most reliable results.
You can find more useful information at https://www.gene-quantification.de/
I would strongly empasize not to use 2^-ΔΔCt method. As this method does not correct for differences in efficiencies between your targets a given Cq does not reflect the initial amounts of targets in your sample. At a given Cq (the inital amount of targets may differ 1-2 log scales due to about 10% difference in efficiency. Other said. At an initial amount of target cDNA your Cq may differ 6-8 if your efficiency is 1.8 instead of 2.
Please use LinReg PCR. In the paper many qPCR methods are compared
J.M. Ruijter Evaluation of qPCR curve analysis methods for reliable biomarker discovery Methods 59 (2013) 32–46
You cannot make duch conclusions if you don’t correct for efficiency of individual qPCR reactions.
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