As far as I know I need at least 1 reference gene, 1 target gene, 1 treated sample and 1 control sample to normalize my qPCR data and obtain -double-deltaCT value. Unlike that in my work, I want to compare gene expression in different body parts of all healthy animals. In that case I don't have any control sample here and it's not possible to have one for such analyze. How can I analyze my qPCR data and obtain -double-deltaCT value without control sample and is there any Excel template for it? Should I leave Livak and Pfaffl method and go for another way?
Thank you,
If you don't need a graph with absolute values of expressions and just relative comparison then you can choose one of the samples as a calibrator (control sample). This control sample will have the value of 1 and the rest of the samples will be compared relative to this one. I would choose as control the sample with the lowest expression.
You need to check if you wish to publish. If yes, I suggest you follow the MIQE guidelines:
Article The MIQE Guidelines: minimum Information for Publication of ...
Without proper designed, data obtained will be useless
If you don't need a graph with absolute values of expressions and just relative comparison then you can choose one of the samples as a calibrator (control sample). This control sample will have the value of 1 and the rest of the samples will be compared relative to this one. I would choose as control the sample with the lowest expression.
As Dragos Peptanariu suggested, select one tissue (the one with highest ct, lowest expression, would make results more readable) and use it as calibrator or control sample (final expression of gene of interest in this tissue 1) and calculate the delta delta ct for all tissues.
I think that whether you want to compare mRNA expression, always you need a reference group. In your case, one of the different body parts is the "control group" and the others have to be referenciated to this I agree with Drago and Alfonso.
Thanks for all your answers,
As many of you suggest I choose one group (highest Ct, lowest expression) as a control group and complete my analyze. Here I attached an excel templete if somebody needs.
Link: Working Paper qPCR Normalization and SE Calculation with Graph, Excel Sheet
Thank you, Mehmet! And thanks for everyone else that helped. I'm in a similar situation now; I want to compare the same mouse tissue in two different ages, with no treatment at all, and I was going crazy because I need to quantify so many genes!
Anyway I'll check the links but you guys already saved me.
I am also stuck in a same situation. But my problem is I am having as much as 80 genes to analysed between tumor tissues matched control and non matched control(tissues from individuals without tumor). In this scenario the I am not sure which one I should consider as 1. because not all the 80 genes in one sample is least. what I want to mean is in some samples some gene is least while in some other sample some other gene is least expressed. So in this case which sample I should choose as 1 or reference?
Thanks...
Hello,
I have a same question as Dipayan, I have 20 genes and two groups of tissue from same organism but with two different treatments so how can I analyse data without control as all these genes have different cq some less and some high.
I would really appreciated if someone could help me with this issue.
Thanks....
To answer to Dipayan and Shirin, I would say that you can actually create an average Ct value as reference.
It`s really well explained on this website (point 3):
https://toptipbio.com/delta-delta-ct-pcr/
Good luck
- Badges
- Science topic