I am looking at the difference in gene expression for a particular target gene and how this changes depending on a specific treatment. I have a house-keeping gene as a reference. I am using the delta-delta Ct calculations to determine this, but am getting results that I can't wrap my head around.
Where I have done the treatment I have gotten a Ct value for the target gene (approx. 33), whereas if I haven't done the treatment I don't get any signal for the target gene. If I follow along the different steps and calculations needed for the delta-delta ct method I end up with fold gene expression that is negative - which suggests a downregulation? But if I get a signal where I have done a treatment, shouldn't the fold gene expression be positive, suggesting up regulation, if there's no signal for the control (untreated one?).
The end value is also extremely small which I also was worried.
Would appreciate any help in clearing this up for me!
When you calculate the delta ct of the untreated which value did you use? If the untreated gives no signal (undetermined) I dont know how you can proceed with the calculation exept it you give it an arbitrary value like 38 ct for exemple. In this case the fold chnage will be positif I think.
Let me know how you did your calculation
To my knowledge, a Ct value of 33 is borderline negative.
Also, you cannot analyze undetectable genes by the ddCt method.
I would try to change the qPCR parameters in order to get Ct values
Hi,
did you use the correct order for ddCq calculatio meaning during substraction to obtain dCq? In addition as said from the others you cannot get a fold change if it is not at all expressed in the control sample.
Regarding the cq values itself: Cq 33 is ok and represents ~ 100 copies of your target in the reaction. However, I would check the PCR efficiency and potentially use more template as this might also lead to a signal in you reference sample (in case you are not limited in material and not already using a high amount of cDNA9.
I am also in a similar situation, just wondering if anyone can clarify my findings. I completed an experiment similar to Labolina. I am looking at the difference in gene expression for a particular target genes and how this changes depending on specific drug treatments, using HPRT as my housekeeping gene and using my positive control of pooled cDNA as my calibrator. Across all my results so far everything has given me a negative ΔΔCt value, which to my knowledge indicates a down regulation, so when I subsequently calculate the expression fold change (2^ -ΔΔCt) I get a 1000 fold down regulation of the GOI in treatments comapred to HPRT. I am right in thinking these assumptions?
Would appreciate any help in clarifying this for me.
Hannah Rowe a negative ddCT gives a positive fold change (RQ value), because of the minus sign in 2^(-ddCT).
Martin Chenal, thank you for getting back to me so quickly, I'm relatively new to PCR so I'm still trying to get to grips with this sensitive technique.
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